Pyrrolobenzodiazepines and conjugates thereof

ABSTRACT

A compound which is either A: 
                         
and salts and solvates thereof, as well as their conjugates with a cell-binding agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/412,138, filed on May 14, 2019, which is a continuation of U.S.application Ser. No. 15/847,308, filed on Dec. 19, 2017, now U.S. Pat.No. 10,335,497, issued on Jul. 2, 2019, which is a continuation of U.S.application Ser. No. 14/051,743, filed on Oct. 11, 2013, now U.S. Pat.No. 9,889,207, issued on Feb. 13, 2018, which claims the benefit of U.S.Provisional Application No. 61/712,928, filed on Oct. 11, 2012, theentire contents of which are incorporated herein by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 868,733 Byte ASCII (Text) file named“2020-04-01_36197-305_SQL_ST25.TXT,” created on Apr. 1, 2020.

The present invention relates to pyrrolobenzodiazepines (PBDs), inparticular pyrrolobenzodiazepines having a labile N10 protecting group,in the form of a linker to a cell binding agent.

BACKGROUND TO THE INVENTION

Pyrrolobenzodiazepines

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965);Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Sincethen, a number of naturally occurring PBDs have been reported, and over10 synthetic routes have been developed to a variety of analogues(Thurston, et al., Chem. Rev. 1994, 433-465 (1994); Antonow, D. andThurston, D. E., Chem. Rev. 2011 111 (4), 2815-2864). Family membersinclude abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667(1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29,93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29,2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97(1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704(1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin(Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin(Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of thegeneral structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine(NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe))at the N10-C11 position which is the electrophilic centre responsiblefor alkylating DNA. All of the known natural products have an(S)-configuration at the chiral C11a position which provides them with aright-handed twist when viewed from the C ring towards the A ring. Thisgives them the appropriate three-dimensional shape for isohelicity withthe minor groove of B-form DNA, leading to a snug fit at the bindingsite (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11(1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237(1986)). Their ability to form an adduct in the minor groove, enablesthem to interfere with DNA processing, hence their use as antitumouragents.

A particularly advantageous pyrrolobenzodiazepine compound is describedby Gregson et al. (Chem. Commun. 1999, 797-798) as compound 1, and byGregson et al. (J. Med. Chem. 2001, 44, 1161-1174) as compound 4a. Thiscompound, also known as SG2000, is shown below:

WO 2007/085930 describes the preparation of dimer PBD compounds havinglinker groups for connection to a cell binding agent, such as anantibody. The linker is present in the bridge linking the monomer PBDunits of the dimer.

The present inventors have described dimer PBD compounds having linkergroups for connection to a cell binding agent, such as an antibody, inWO 2011/130598. The linker in these compounds is attached to one of theavailable N10 positions, and are generally cleaved by action of anenzyme on the linker group.

Antibody-Drug Conjugates

Antibody therapy has been established for the targeted treatment ofpatients with cancer, immunological and angiogenic disorders (Carter, P.(2006) Nature Reviews Immunology 6:343-357). The use of antibody-drugconjugates (ADC), i.e. immunoconjugates, for the local delivery ofcytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumorcells in the treatment of cancer, targets delivery of the drug moiety totumors, and intracellular accumulation therein, whereas systemicadministration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells (Xie et al (2006)Expert. Opin. Biol. Ther. 6(3):281-291; Kovtun et al (2006) Cancer Res.66(6):3214-3121; Law et al (2006) Cancer Res. 66(4):2328-2337; Wu et al(2005) Nature Biotech. 23(9):1137-1145; Lambert J. (2005) Current Opin.in Pharmacol. 5:543-549; Hamann P. (2005) Expert Opin. Ther. Patents15(9):1087-1103; Payne, G. (2003) Cancer Cell 3:207-212; Trail et al(2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos(1999) Anticancer Research 19:605-614).

Maximal efficacy with minimal toxicity is sought thereby. Efforts todesign and refine ADC have focused on the selectivity of monoclonalantibodies (mAbs) as well as drug mechanism of action, drug-linking,drug/antibody ratio (loading), and drug-releasing properties (Junutula,et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood114(13):2721-2729; U.S. Pat. Nos. 7,521,541; 7,723,485; WO2009/052249;McDonagh (2006) Protein Eng. Design & Sel. 19(7): 299-307; Doronina etal (2006) Bioconj. Chem. 17:114-124; Erickson et al (2006) Cancer Res.66(8):1-8; Sanderson et al (2005) Clin. Cancer Res. 11:843-852; Jeffreyet al (2005) J. Med. Chem. 48:1344-1358; Hamblett et al (2004) Clin.Cancer Res. 10:7063-7070). Drug moieties may impart their cytotoxic andcytostatic effects by mechanisms including tubulin binding, DNA binding,proteasome and/or topoisomerase inhibition. Some cytotoxic drugs tend tobe inactive or less active when conjugated to large antibodies orprotein receptor ligands.

The present inventors have developed particular PBD dimers with linkinggroups for the formation of PBD conjugates with cell binding agents, andin particular PBD antibody conjugates.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides compound A:

and salts of solvates thereof.

WO 2011/130598 discloses compound 80:

Compound A differs from this by comprising an iodoacetamide group forlinking to the cell binding agent. This group may offer advantages overcompound 80 with regards to its stability when bound to a cell bindingagent (see below). The malemide group in compound 80 can undergo aretro-Michael reaction, becoming unconjugated from the cell bindingagent, and thus vunerable to scavenging by other thiol containingbiological molecules, such as albumin and glutathione. Suchunconjugation cannot occur with compound A. Also, the iodoacetamidegroup may avoid other unwanted side reactions.

In a second aspect, the present invention provides compound B:

and salts and solvates thereof.

Compound B differs from previously disclosed PBD dimers with a druglinker having a C2-3 endo-double bond, by having a smaller, lesslipophilic C2 substituent, e.g. 4F-phenyl, propylene. As such, theconjugates of compound B (see below) are less likely to aggregate oncesynthesised. Such aggregation of conjugates can be measured by Sizeexclusion chromatography (SEC).

Both compound A and B have two sp² centres in each C-ring, which mayallow for stronger binding in the minor groove of DNA, than forcompounds with only one sp² centre in each C-ring.

A third aspect of the present invention provides a conjugate of formulaConjA:

or ConjB:

where CBA represents a cell binding agent. The link to the moiety shownis via a free S (active thiol) on the cell binding agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect on mean tumour volume in groups of 10 mice dosedwith ADC1A at 0.3 (green) or 1.0 mg/kg (red) compared to vehicle (black)or naked Ig (blue) controls.

FIG. 2 shows the effect on mean tumour volume in groups of 10 mice dosedwith ADC1B at 0.3 (light blue) or 1.0 mg/kg (green) compared to vehicle(black) or naked Ig (dark blue) controls.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a PBD dimer with a linker connectedthrough the N10 position on one of the PBD moieties suitable for forminga PBD dimer conjugated via the linker to a cell binding agent.

The present invention is suitable for use in providing a PBD compound toa preferred site in a subject. The conjugate allows the release of anactive PBD compound that does not retain any part of the linker. Thereis no stub present that could affect the reactivity of the PBD compound.Thus ConjA would release the compound RelA:

and ConjB would release the compound RelB:

A further aspect of the present invention is the compound RelB, andsalts and solvates thereof.

The specified link between the PBD dimer and the cell binding agent,e.g. antibody, in the present invention is preferably stableextracellularly. Before transport or delivery into a cell, theantibody-drug conjugate (ADC) is preferably stable and remains intact,i.e. the antibody remains linked to the drug moiety. The linkers arestable outside the target cell and may be cleaved at some efficaciousrate inside the cell. An effective linker will: (i) maintain thespecific binding properties of the antibody; (ii) allow intracellulardelivery of the conjugate or drug moiety; (iii) remain stable andintact, i.e. not cleaved, until the conjugate has been delivered ortransported to its targeted site; and (iv) maintain a cytotoxic,cell-killing effect or a cytostatic effect of the PBD drug moiety.Stability of the ADC may be measured by standard analytical techniquessuch as mass spectroscopy, HPLC, and the separation/analysis techniqueLC/MS.

Delivery of the compounds of formulae RelA or RelB is achieved at thedesired activation site of the conjugates of formulae ConjA or ConjB bythe action of an enzyme, such as cathepsin, on the linking group, and inparticular on the valine-alanine dipeptide moiety.

Cell Binding Agent

A cell binding agent may be of any kind, and include peptides andnon-peptides. These can include antibodies or a fragment of an antibodythat contains at least one binding site, lymphokines, hormones, hormonemimetics, vitamins, growth factors, nutrient-transport molecules, or anyother cell binding molecule or substance.

Peptides

In one embodiment, the cell binding agent is a linear or cyclic peptidecomprising 4-30, preferably 6-20, contiguous amino acid residues. Inthis embodiment, it is preferred that one cell binding agent is linkedto one monomer or dimer pyrrolobenzodiazepine compound.

In one embodiment the cell binding agent comprises a peptide that bindsintegrin α_(v)β₆. The peptide may be selective for α_(v)β₆ over XYS.

In one embodiment the cell binding agent comprises the A20FMDV-Cyspolypeptide. The A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC(SEQ ID NO: 1). Alternatively, a variant of the A20FMDV-Cys sequence maybe used wherein one, two, three, four, five, six, seven, eight, nine orten amino acid residues are substituted with another amino acid residue.Furthermore, the polypeptide may have the sequence NAVXXXXXXXXXXXXXXXRTC(SEQ ID NO: 2).

Antibodies

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein generated by theimmune system that is capable of recognizing and binding to a specificantigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen mayhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin can be of anytype (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Theimmunoglobulins can be derived from any species, including human,murine, or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and scFv fragments;diabodies; linear antibodies; fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR (complementarydetermining region), and epitope-binding fragments of any of the abovewhich immunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens, single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al (1991) Nature, 352:624-628; Markset al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carryinga fully human immunoglobulin system (Lonberg (2008) Curr. Opinion20(4):450-459).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodiesinclude “primatized” antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g. OldWorld Monkey or Ape) and human constant region sequences.

An “intact antibody” herein is one comprising a VL and VH domains, aswell as a light chain constant domain (CL) and heavy chain constantdomains, CH1, CH2 and CH3. The constant domains may be native sequenceconstant domains (e.g. human native sequence constant domains) or aminoacid sequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

Humanisation

Techniques to reduce the in vivo immunogenicity of a non-human antibodyor antibody fragment include those termed “humanisation”.

A “humanized antibody” refers to a polypeptide comprising at least aportion of a modified variable region of a human antibody wherein aportion of the variable region, preferably a portion substantially lessthan the intact human variable domain, has been substituted by thecorresponding sequence from a non-human species and wherein the modifiedvariable region is linked to at least another part of another protein,preferably the constant region of a human antibody. The expression“humanized antibodies” includes human antibodies in which one or morecomplementarity determining region (“CDR”) amino acid residues and/orone or more framework region (“FW” or “FR”) amino acid residues aresubstituted by amino acid residues from analogous sites in rodent orother non-human antibodies. The expression “humanized antibody” alsoincludes an immunoglobulin amino acid sequence variant or fragmentthereof that comprises an FR having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. Or, looked at another way, a humanized antibody is ahuman antibody that also contains selected sequences from non-human(e.g. murine) antibodies in place of the human sequences. A humanizedantibody can include conservative amino acid substitutions ornon-natural residues from the same or different species that do notsignificantly alter its binding and/or biologic activity. Suchantibodies are chimeric antibodies that contain minimal sequence derivedfrom non-human immunoglobulins.

There are a range of humanisation techniques, including ‘CDR grafting’,‘guided selection’, ‘deimmunization’, ‘resurfacing’ (also known as‘veneering’), ‘composite antibodies’, ‘Human String ContentOptimisation’ and framework shuffling.

CDR Grafting

In this technique, the humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient antibody are replaced by residues from aCDR of a non-human species (donor antibody) such as mouse, rat, camel,bovine, goat, or rabbit having the desired properties (in effect, thenon-human CDRs are ‘grafted’ onto the human framework). In someinstances, framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues (this may happen when,for example, a particular FR residue has significant effect on antigenbinding).

Furthermore, humanized antibodies can comprise residues that are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. Thus, in general, a humanized antibody willcomprise all of at least one, and in one aspect two, variable domains,in which all or all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), or that of a human immunoglobulin.

Guided Selection

The method consists of combining the V_(H) or V_(L) domain of a givennon-human antibody specific for a particular epitope with a human V_(H)or V_(L) library and specific human V domains are selected against theantigen of interest. This selected human VH is then combined with a VLlibrary to generate a completely human VH×VL combination. The method isdescribed in Nature Biotechnology (N.Y.) 12, (1994) 899-903.

Composite Antibodies

In this method, two or more segments of amino acid sequence from a humanantibody are combined within the final antibody molecule. They areconstructed by combining multiple human VH and VL sequence segments incombinations which limit or avoid human T cell epitopes in the finalcomposite antibody V regions. Where required, T cell epitopes arelimited or avoided by, exchanging V region segments contributing to orencoding a T cell epitope with alternative segments which avoid T cellepitopes. This method is described in US 2008/0206239 A1.

Deimmunization

This method involves the removal of human (or other second species)T-cell epitopes from the V regions of the therapeutic antibody (or othermolecule). The therapeutic antibodies V-region sequence is analysed forthe presence of MHC class II-binding motifs by, for example, comparisonwith databases of MHC-binding motifs (such as the “motifs” databasehosted at www.wehi.edu.au). Alternatively, MHC class II-binding motifsmay be identified using computational threading methods such as thosedevised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)); in thesemethods, consecutive overlapping peptides from the V-region sequencesare testing for their binding energies to MHC class II proteins. Thisdata can then be combined with information on other sequence featureswhich relate to successfully presented peptides, such as amphipathicity,Rothbard motifs, and cleavage sites for cathepsin B and other processingenzymes.

Once potential second species (e.g. human) T-cell epitopes have beenidentified, they are eliminated by the alteration of one or more aminoacids. The modified amino acids are usually within the T-cell epitopeitself, but may also be adjacent to the epitope in terms of the primaryor secondary structure of the protein (and therefore, may not beadjacent in the primary structure). Most typically, the alteration is byway of substitution but, in some circumstances amino acid addition ordeletion will be more appropriate.

All alterations can be accomplished by recombinant DNA technology, sothat the final molecule may be prepared by expression from a recombinanthost using well established methods such as Site Directed Mutagenesis.However, the use of protein chemistry or any other means of molecularalteration is also possible.

Resurfacing

This method involves:

-   -   (a) determining the conformational structure of the variable        region of the non-human (e.g. rodent) antibody (or fragment        thereof) by constructing a three-dimensional model of the        non-human antibody variable region;    -   (b) generating sequence alignments using relative accessibility        distributions from x-ray crystallographic structures of a        sufficient number of non-human and human antibody variable        region heavy and light chains to give a set of heavy and light        chain framework positions wherein the alignment positions are        identical in 98% of the sufficient number of non-human antibody        heavy and light chains;    -   (c) defining for the non-human antibody to be humanized, a set        of heavy and light chain surface exposed amino acid residues        using the set of framework positions generated in step (b);    -   (d) identifying from human antibody amino acid sequences a set        of heavy and light chain surface exposed amino acid residues        that is most closely identical to the set of surface exposed        amino acid residues defined in step (c), wherein the heavy and        light chain from the human antibody are or are not naturally        paired;    -   (e) substituting, in the amino acid sequence of the non-human        antibody to be humanized, the set of heavy and light chain        surface exposed amino acid residues defined in step (c) with the        set of heavy and light chain surface exposed amino acid residues        identified in step (d);    -   (f) constructing a three-dimensional model of the variable        region of the non-human antibody resulting from the substituting        specified in step (e);    -   (g) identifying, by comparing the three-dimensional models        constructed in steps (a) and (f), any amino acid residues from        the sets identified in steps (c) or (d), that are within 5        Angstroms of any atom of any residue of the complementarity        determining regions of the non-human antibody to be humanized;        and    -   (h) changing any residues identified in step (g) from the human        to the original non-human amino acid residue to thereby define a        non-human antibody humanizing set of surface exposed amino acid        residues; with the proviso that step (a) need not be conducted        first, but must be conducted prior to step (g).

Superhumanization

The method compares the non-human sequence with the functional humangermline gene repertoire. Those human genes encoding canonicalstructures identical or closely related to the non-human sequences areselected. Those selected human genes with highest homology within theCDRs are chosen as FR donors. Finally, the non-human CDRs are graftedonto these human FRs. This method is described in patent WO 2005/079479A2.

Human String Content Optimization

This method compares the non-human (e.g. mouse) sequence with therepertoire of human germline genes and the differences are scored asHuman String Content (HSC) that quantifies a sequence at the level ofpotential MHC/T-cell epitopes. The target sequence is then humanized bymaximizing its HSC rather than using a global identity measure togenerate multiple diverse humanized variants (described in MolecularImmunology, 44, (2007) 1986-1998).

Framework Shuffling

The CDRs of the non-human antibody are fused in-frame to cDNA poolsencompassing all known heavy and light chain human germline geneframeworks. Humanised antibodies are then selected by e.g. panning ofthe phage displayed antibody library. This is described in Methods 36,43-60 (2005).

Examples of cell binding agents include those agents described for usein WO 2007/085930, which is incorporated herein.

Tumour-associate antigens and cognate antibodies for use in embodimentsof the present invention are listed below.

Tumor-Associated Antigens and Cognate Antibodies

(1) BMPR1B (bone morphogenetic protein receptor-type IB)

Nucleotide

Genbank accession no. NM_001203

Genbank version no. NM_001203.2 GI:169790809 (SEQ ID NO: 45)

Genbank record update date: Sep. 23, 2012 02:06 PM

Polypeptide

Genbank accession no. NP_001194

Genbank version no. NP_001194.1 GI:4502431 (SEQ ID NO: 46)

Genbank record update date: Sep. 23, 2012 02:06 PM

CROSS-REFERENCES

-   ten Dijke, P., et al Science 264 (5155): 101-104 (1994), Oncogene 14    10 (11):1377-1382 (1997)); WO2004/063362 (Claim 2); WO2003/042661    (Claim 12);-   US2003/134790-A1 (Page 38-39); WO2002/102235 (Claim 13; Page 296);    WO2003/055443 (Page 91-92); WO2002/99122 (Example 2; Page 528-530);    WO2003/029421 (Claim 6); WO2003/024392 (Claim 2; FIG. 112);    WO2002/98358 (Claim 1; Page 183); WO2002/54940 (Page 100-101);    WO2002/59377(Page 349-350); WO2002/30268 (Claim 27; Page 376); 15    WO2001/48204 (Example; FIG. 4); NP_001194 bone morphogenetic protein    receptor, type IB/pid=NP_001194.1; MIM:603248; AY065994

(2) E16 (LAT1, SLC7A5)

Nucleotide

Genbank accession no. NM_003486

Genbank version no. NM_003486.5 GI:71979931 (SEQ ID NO: 47)

Genbank record update date: Jun. 27, 2012 12:06 PM

Polypeptide

Genbank accession no. NP 003477

Genbank version no. NP_003477.4 GI:71979932 (SEQ ID NO: 48)

Genbank record update date: Jun. 27, 2012 12:06 PM

CROSS REFERENCES

-   Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395    (6699):288-291 (1998), Gaugitsch, H. W., et 20 al (1992) J. Biol.    Chem. 267 (16):11267-11273); WO2004/048938 (Example 2);    WO2004/032842 (Example IV); WO2003/042661 (Claim 12); WO2003/016475    (Claim 1); WO2002/78524 (Example 2); WO2002/99074 (Claim 19; Page    127-129); WO2002/86443 (Claim 27; Pages 222, 393); WO2003/003906    (Claim 10; Page 293); WO2002/64798 (Claim 33; Page 93-95);    WO2000/14228 (Claim 5; Page 133-136); US2003/224454 (FIG. 3); 25    WO2003/025138 (Claim 12; Page 150); NP_003477 solute carrier family    7 (cationic amino acid transporter, y+system), member    5/pid=NP_003477.3-Homo sapiens; MIM:600182; NM_015923.

(3) STEAP1 (six transmembrane epithelial antigen of prostate)

Nucleotide

Genbank accession no. NM_012449

Genbank version no. NM_012449.2 GI:22027487 (SEQ ID NO: 49)

Genbank record update date: Sep. 9, 2012 02:57 PM

Polypeptide

Genbank accession no. NP 036581

Genbank version no. NP_036581.1 GI:9558759 (SEQ ID NO: 50)

Genbank record update date: Sep. 9, 2012 02:57 PM

CROSS REFERENCES

-   Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R. S., et al (1999)    Proc. Natl. Acad. Sci. U.S.A. 96 (25):14523-14528); WO2004/065577    (Claim 6); WO2004/027049 (FIG. 1L); EP1394274 (Example 11);    WO2004/016225 (Claim 2); WO2003/042661 (Claim 12); US2003/157089    (Example 5); US2003/185830 (Example 5); US2003/064397 (FIG. 2);    WO2002/89747 (Example 5; Page 618-619); WO2003/022995 (Example 9;    FIG. 13A, 35 Example 53; Page 173, Example 2; FIG. 2A); six    transmembrane epithelial antigen of the prostate; MIM:604415.

(4) 0772P (CA125, MUC16)

Nucleotide

Genbank accession no. AF361486

Genbank version no. AF361486.3 GI:34501466 (SEQ ID NO: 51)

Genbank record update date: Mar. 11, 2010 07:56 AM

Polypeptide

Genbank accession no. AAK74120

Genbank version no. AAK74120.3 GI:34501467 (SEQ ID NO: 52)

Genbank record update date: Mar. 11, 2010 07:56 AM

CROSS REFERENCES

-   J. Biol. Chem. 276 (29):27371-27375 (2001)); WO2004/045553 (Claim    14); WO2002/92836 (Claim 6; FIG. 12); WO2002/83866 (Claim 15; Page    116-121); US2003/124140 (Example 16); GI:34501467;

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin)

Nucleotide

Genbank accession no. NM_005823

Genbank version no. NM_005823.5 GI:293651528 (SEQ ID NO: 53)

Genbank record update date: Sep. 2, 2012 01:47 PM

Polypeptide

Genbank accession no. NP 005814

Genbank version no. NP_005814.2 GI:53988378 (SEQ ID NO: 54)

Genbank record update date: Sep. 2, 2012 01:47 PM

CROSS REFERENCES

-   Yamaguchi, N., et al Biol. Chem. 269 (2), 805-808 (1994), Proc.    Natl. Acad. Sci. U.S.A. 96 (20):11531-11536 (1999), Proc. Natl.    Acad. Sci. U.S.A. 93 10 (1):136-140 (1996), J. Biol. Chem. 270    (37):21984-21990 (1995)); WO2003/101283 (Claim 14); (WO2002/102235    (Claim 13; Page 287-288); WO2002/101075 (Claim 4; Page 308-309);    WO2002/71928 (Page 320-321); WO94/10312 (Page 52-57); IM:601051.

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type ii sodium-dependent phosphate transporter 3b)

Nucleotide

Genbank accession no. NM_006424

Genbank version no. NM_006424.2 GI:110611905 (SEQ ID NO: 55)

Genbank record update date: Jul. 22, 2012 03:39 PM

Polypeptide

Genbank accession no. NP 006415

Genbank version no. NP_006415.2 GI:110611906 (SEQ ID NO: 56)

Genbank record update date: Jul. 22, 2012 03:39 PM

CROSS REFERENCES

-   J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284    (1999), Feild, J. A., et al (1999) Biochem. Biophys. Res. Commun.    258 (3):578-582); WO2004/022778 (Claim 2); EP1394274 (Example 11);    WO2002/102235 (Claim 13; Page 20 326); EP0875569 (Claim 1; Page    17-19); WO2001/57188 (Claim 20; Page 329); WO2004/032842 (Example    IV); WO2001/75177 (Claim 24; Page 139-140); MIM:604217.

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, 25 sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B)

Nucleotide

Genbank accession no. AB040878

Genbank version no. AB040878.1 GI:7959148 (SEQ ID NO: 57)

Genbank record update date: Aug. 2, 2006 05:40 PM

Polypeptide

Genbank accession no. BAA95969

Genbank version no. BAA95969.1 GI:7959149 (SEQ ID NO: 58)

Genbank record update date: Aug. 2, 2006 05:40 PM

CROSS REFERENCES

-   Nagase T., et al (2000) DNA Res. 7 (2):143-150); WO2004/000997    (Claim 1); WO2003/003984 (Claim 1); WO2002/06339 (Claim 1; Page 50);    WO2001/88133 (Claim 1; Page 41-43, 48-58); WO2003/054152 (Claim 20);    WO2003/101400 (Claim 11); Accession: 30 Q9P283; Genew; HGNC:10737

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene)

Nucleotide

Genbank accession no. AY358628

Genbank version no. AY358628.1 GI:37182377 (SEQ ID NO: 59)

Genbank record update date: Dec. 1, 2009 04:15 AM

Polypeptide

Genbank accession no. AAQ88991

Genbank version no. AAQ88991.1 GI:37182378 (SEQ ID NO: 60)

Genbank record update date: Dec. 1, 2009 04:15 AM

CROSS REFERENCES

-   Ross et al (2002) Cancer Res. 62:2546-2553; US2003/129192 (Claim 2);    US2004/044180 (Claim 12); US2004/044179 35 (Claim 11); US2003/096961    (Claim 11); US2003/232056 (Example 5); WO2003/105758 16 (Claim 12);    US2003/206918 (Example 5); EP1347046 (Claim 1); WO2003/025148 (Claim    20); GI:37182378.

(9) ETBR (Endothelin type B receptor)

Nucleotide

Genbank accession no. AY275463

Genbank version no. AY275463.1 GI:30526094 (SEQ ID NO: 61)

Genbank record update date: Mar. 11, 2010 02:26 AM

Polypeptide

Genbank accession no. AAP32295

Genbank version no. AAP32295.1 GI:30526095 (SEQ ID NO: 62)

Genbank record update date: Mar. 11, 2010 02:26 AM

CROSS REFERENCES

-   Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991;    Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255, 1991;    Arai H., et al Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J.    Biol. Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al    Biochem. Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N.    A., et al J. Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J.    Cardiovasc. Pharmacol. 20, sl-S4, 1992; Tsutsumi M., et al Gene 228,    43-49, 1999; Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A.    99, 16899-16903, 2002; Bourgeois C., et al J. Clin. Endocrinol.    Metab. 82, 3116-3123, 1997;-   Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J. B.,    et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et al    Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Cell    79, 1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-15    2409, 1995; Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996;    Amiel J., et al Hum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W.,    et al Nat. Genet. 12, 445-447, 1996; Svensson P. J., et al Hum.    Genet. 103, 145-148, 1998; Fuchs S., et al Mol. Med. 7, 115-124,    2001; Pingault V., et al (2002) Hum. Genet. 111, 198-206;    WO2004/045516 (Claim 1); WO2004/048938 (Example 2); WO2004/040000    (Claim 151); WO2003/087768 (Claim 1); 20 WO2003/016475 (Claim 1);    WO2003/016475 (Claim 1); WO2002/61087 (FIG. 1); WO2003/016494 (FIG.    6); WO2003/025138 (Claim 12; Page 144); WO2001/98351 (Claim 1; Page    124-125); EP0522868 (Claim 8; FIG. 2); WO2001/77172 (Claim 1; Page    297-299); US2003/109676; U.S. Pat. No. 6,518,404 (FIG. 3); U.S. Pat.    No. 5,773,223 (Claim 1a; Col 31-34); WO2004/001004.

(10) MSG783 (RNF124, hypothetical protein FLJ20315)

Nucleotide

Genbank accession no. NM_017763

Genbank version no. NM_017763.4 GI:167830482 (SEQ ID NO: 63)

Genbank record update date: Jul. 22, 2012 12:34 AM

Polypeptide

Genbank accession no. NP 060233

Genbank version no. NP_060233.3 GI:56711322 (SEQ ID NO: 64)

Genbank record update date: Jul. 22, 2012 12:34 AM

CROSS REFERENCES

-   WO2003/104275 (Claim 1); WO2004/046342 (Example 2); WO2003/042661    (Claim 12); WO2003/083074 (Claim 14; Page 61); WO2003/018621 (Claim    1); WO2003/024392 (Claim 2; FIG. 93); WO2001/66689 (Example 6);    LocusiD:54894.

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostatecancer associated gene 1, prostate cancer associated protein 1, sixtransmembrane epithelial antigen of prostate 2, six transmembraneprostate protein)

Nucleotide

Genbank accession no. AF455138

Genbank version no. AF455138.1 GI:22655487 (SEQ ID NO: 65)

Genbank record update date: Mar. 11, 2010 01:54 AM

Polypeptide

Genbank accession no. AAN04080

Genbank version no. AAN04080.1 GI:22655488 (SEQ ID NO: 66)

Genbank record update date: Mar. 11, 2010 01:54 AM

CROSS REFERENCES

-   Lab. Invest. 82 (11):1573-1582 (2002)); WO2003/087306; US2003/064397    (Claim 1; FIG. 1); WO2002/72596 (Claim 13; Page 54-55); WO2001/72962    (Claim 1; FIG. 4B); 35 WO2003/104270 (Claim 11); WO2003/104270    (Claim 16); US2004/005598 (Claim 22); WO2003/042661 (Claim 12);    US2003/060612 (Claim 12; FIG. 10); WO2002/26822 (Claim 23; FIG. 2);    WO2002/16429 (Claim 12; FIG. 10); GI:22655488.

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation 5 channel, subfamily M, member 4)

Nucleotide

Genbank accession no. NM_017636

Genbank version no. NM_017636.3 GI:304766649 (SEQ ID NO: 67)

Genbank record update date: Jun. 29, 2012 11:27 AM

Polypeptide

Genbank accession no. NP 060106

Genbank version no. NP_060106.2 GI:21314671 (SEQ ID NO: 68)

Genbank record update date: Jun. 29, 2012 11:27 AM

CROSS REFERENCES

-   Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697    (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278    (33):30813-30820 (2003)); US2003/143557 (Claim 4); WO2000/40614    (Claim 14; Page 100-103); WO2002/10382 (Claim 1; FIG. 9A);    WO2003/042661 (Claim 12); WO2002/30268 (Claim 27; Page 391);    US2003/219806 (Claim 4); WO2001/62794 (Claim 10 14; FIG. 1A-D);    MIM:606936.

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGFI, teratocarcinoma-derivedgrowth factor)

Nucleotide

Genbank accession no. NM_003212

Genbank version no. NM_003212.3 GI:292494881 (SEQ ID NO: 69)

Genbank record update date: Sep. 23, 2012 02:27 PM

Polypeptide

Genbank accession no. NP 003203

Genbank version no. NP_003203.1 GI:4507425 (SEQ ID NO: 70)

Genbank record update date: Sep. 23, 2012 02:27 PM

CROSS REFERENCES

Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.Genet. 49 (3):555-565 (1991)); US2003/224411 (Claim 1); WO2003/083041(Example 1); WO2003/034984 (Claim 12); WO2002/88170 (Claim 2; Page52-53); WO2003/024392 (Claim 2; FIG. 58); WO2002/16413 (Claim 1; Page94-95, 105); WO2002/22808 (Claim 2; FIG. 1); U.S. Pat. No. 5,854,399(Example 2; Col 17-18); U.S. Pat. No. 5,792,616 (FIG. 2); MIM:187395.

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs. 73792)

Nucleotide

Genbank accession no M26004

Genbank version no. M26004.1 GI:181939 (SEQ ID NO: 71)

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA35786

Genbank version no. AAA35786.1 GI:181940 (SEQ ID NO: 72)

Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS REFERENCES

-   Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J.,    et al J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Natl.    Acad. Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol.    35, 1025-1031, 1998; Weis J. J., et al Proc. Natl. Acad. Sci. U.S.A.    83, 5639-5643, 1986; Sinha S. K., et al (1993) J. Immunol. 150,    5311-5320; WO2004/045520 (Example 4); US2004/005538 (Example 1);    WO2003/062401 (Claim 9); WO2004/045520 (Example 4); WO91/02536    (FIGS. 9.1-9.9); WO2004/020595 (Claim 1); Accession: P20023; Q13866;    Q14212; EMBL; M26004; AAA35786.1.

(15) CD79b (CD79ß, CD7913, IGb (immunoglobulin-associated beta), B29)

Nucleotide

Genbank accession no NM_000626

Genbank version no. NM_000626.2 GI:90193589 (SEQ ID NO: 73)

Genbank record update date: Jun. 26, 2012 01:53 PM

Polypeptide

Genbank accession no. NP 000617

Genbank version no. NP_000617.1 GI:11038674 (SEQ ID NO: 74)

Genbank record update date: Jun. 26, 2012 01:53 PM

CROSS REFERENCES

-   Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126-4131, Blood (2002)    100 (9):3068-3076, Muller et al (1992) Eur. J. Immunol. 22    (6):1621-1625); WO2004/016225 (claim 2, FIG. 140); WO2003/087768,    US2004/101874 (claim 1, page 102); WO2003/062401 (claim 9);    WO2002/78524 (Example 2); US2002/150573 (claim 35 5, page 15); U.S.    Pat. No. 5,644,033; WO2003/048202 (claim 1, pages 306 and 309); WO    99/58658, U.S. Pat. No. 6,534,482 (claim 13, FIG. 17A/B);    WO2000/55351 (claim 11, pages 1145-1146); MIM:147245

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 5 1a), SPAP1B, SPAP1C)

Nucleotide

Genbank accession no NM_030764

Genbank version no. NM_030764.3 GI:227430280 (SEQ ID NO: 75)

Genbank record update date: Jun. 30, 2012 12:30 AM

Polypeptide

Genbank accession no. NP_110391

Genbank version no. NP_110391.2 GI:19923629 (SEQ ID NO: 76)

Genbank record update date: Jun. 30, 2012 12:30 AM

CROSS REFERENCES

-   AY358130); Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54    (2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad.    Sci. U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al (2001)    Biochem. Biophys. Res. Commun. 280 (3):768-775; WO2004/016225 (Claim    2); WO2003/077836; WO2001/38490 (Claim 5; FIGS. 18D-1-18D-2);    WO2003/097803 (Claim 12);-   10 WO2003/089624 (Claim 25): MIM:606509.

(17) HER2 (ErbB2)

Nucleotide

Genbank accession no M11730

Genbank version no. M11730.1 GI:183986 (SEQ ID NO: 77)

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA75493

Genbank version no. AAA75493.1 GI:306840 (SEQ ID NO: 78)

Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS REFERENCES

-   Coussens L., et al Science (1985) 230(4730):1132-1139); Yamamoto T.,    et al Nature 319, 230-234, 1986; Semba K., et al Proc. Natl. Acad.    Sci. U.S.A. 82, 6497-6501, 1985; Swiercz J. M., et al J. Cell Biol.    165, 869-15 880, 2004; Kuhns J. J., et al J. Biol. Chem. 274,    36422-36427, 1999; Cho H.-S., et al Nature 421, 756-760, 2003;    Ehsani A., et al (1993) Genomics 15, 426-429; WO2004/048938 (Example    2); WO2004/027049 (FIG. 11); WO2004/009622; WO2003/081210;-   WO2003/089904 (Claim 9); WO2003/016475 (Claim 1); US2003/118592;    WO2003/008537 (Claim 1); WO2003/055439 (Claim 29; FIG. 1A-B);    WO2003/025228 (Claim 37; FIG. 5C); 20 WO2002/22636 (Example 13; Page    95-107); WO2002/12341 (Claim 68; FIG. 7); WO2002/13847 (Page 71-74);    WO2002/14503 (Page 114-117); WO2001/53463 (Claim 2; Page 41-46);    WO2001/41787 (Page 15); WO2000/44899 (Claim 52; FIG. 7);    WO2000/20579 (Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim 3;    Col 31-38); WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7);    WO2004/043361 (Claim 7); WO2004/022709; WO2001/00244 25 (Example 3;    FIG. 4); Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761;    AAA35808.1

Antibodies

Abbott: US20110177095

-   -   For example, an antibody comprising CDRs having overall at least        80% sequence identity to CDRs having amino acid sequences of SEQ        ID NO:3 (CDR-H1), SEQ ID NO:4 (CDR-H2), SEQ ID NO:5 (CDR-H3),        SEQ ID NO:104 and/or SEQ ID NO:6 (CDR-L1), SEQ ID NO:7 (CDR-L2),        and SEQ ID NO:8 (CDR-L3), wherein the anti-HER2 antibody or        anti-HER2 binding fragment has reduced immunogenicity as        compared to an antibody having a VH of SEQ ID NO:1 and a VL of        SEQ ID NO:2.

Biogen: US20100119511

-   -   For example, ATCC accession numbers: PTA-10355, PTA-10356,        PTA-10357, PTA-10358    -   For example, a purified antibody molecule that binds to HER2        comprising a all six CDR's from an antibody selected from the        group consisting of BIIB71F10 (SEQ ID NOs:11, 13), BllB69A09        (SEQ ID NOs:15, 17); BllB67F10 (SEQ ID NOs:19, 21); BIIB67F11        (SEQ ID NOs:23, 25), BllB66A12 (SEQ ID NOs:27, 29), BllB66C01        (SEQ ID NOs:31, 33), BllB65C10 (SEQ ID NOs:35, 37), BllB65H09        (SEQ ID NOs:39, 41) and BllB65B03 (SEQ ID NOs:43, 45), or CDRs        which are identical or which have no more than two alterations        from said CDRs.

Herceptin (Genentech)—U.S. Pat. No. 6,054,297; ATCC accession no.CRL-10463 (Genentech)

Pertuzumab (Genentech)

-   -   US20110117097        -   for example, see SEQ IDs No. 15&16, SEQ IDs No. 17&18, SEQ            IDs No. 23&24 & ATCC accession numbers HB-12215, HB-12216,            CRL 10463, HB-12697.    -   US20090285837    -   US20090202546        -   for example, ATCC accession numbers: HB-12215, HB-12216, CRL            10463, HB-12698.    -   US20060088523        -   for example, ATCC accession numbers: HB-12215, HB-12216        -   for example, an antibody comprising the variable light and            variable heavy amino acid sequences in SEQ ID Nos. 3 and 4,            respectively.        -   for example, an antibody comprising a light chain amino acid            sequence selected from SEQ ID No. 15 and 23, and a heavy            chain amino acid sequence selected from SEQ ID No. 16 and 24    -   US20060018899        -   for example, ATCC accession numbers: (7C2) HB-12215, (7F3)            HB-12216, (4D5) CRL-10463, (2C4) HB-12697.        -   for example, an antibody comprising the amino acid sequence            in SEQ ID No. 23, or a deamidated and/or oxidized variant            thereof.    -   US2011/0159014        -   for example, an antibody having a light chain variable            domain comprising the hypervariable regions of SEQ ID NO:            1”.        -   For example, an antibody having a heavy chain variable            domain comprising the hypervariable regions of SEQ ID NO: 2.    -   US20090187007

Glycotope: TrasGEX antibody http://www.glycotope.com/pipeline

-   -   For example, see International Joint Cancer Institute and        Changhai Hospital Cancer Cent: HMTI-Fc Ab—Gao J., et al BMB Rep.        2009 Oct. 31; 42(10):636-41.

Symphogen: US20110217305

-   Union Stem Cell &Gene Engineering, China—Liu HQ., et al Xi Bao Yu    Fen Zi Mian YiXue Za Zhi. 2010 May; 26(5):456-8.

(18) NCA (CEACAM6)

Nucleotide

Genbank accession no M18728

Genbank version no. M18728.1 GI:189084 (SEQ ID NO: 79)

Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA59907

Genbank version no. AAA59907.1 GI:189085 (SEQ ID NO: 80)

Genbank record update date: Jun. 23, 2010 08:48 AM

CROSS REFERENCES

-   Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al    Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L.,    et al Proc. Natl. Acad. Sci. U.S.A. 99:16899-16903, 2002;    WO2004/063709; EP1439393 (Claim 7); WO2004/044178 (Example 4);    WO2004/031238; WO2003/042661 (Claim 12); WO2002/78524 (Example 2);    WO2002/86443 (Claim 27; Page 427); WO2002/60317 (Claim 2);    Accession: P40199; Q14920; EMBL; M29541; AAA59915.1.

EMBL; M18728.

(19) MDP (DPEP1)

Nucleotide

Genbank accession no BC017023

Genbank version no. BC017023.1 GI:16877538 (SEQ ID NO: 81)

Genbank record update date: Mar. 6, 2012 01:00 PM

Polypeptide

Genbank accession no. AAH17023

Genbank version no. AAH17023.1 GI:16877539 (SEQ ID NO: 82)

Genbank record update date: Mar. 6, 2012 01:00 PM

CROSS REFERENCES

-   Proc. Natl. Acad. Sci. U.S.A. 99 (26):16899-16903 (2002));    WO2003/016475 (Claim 1); WO2002/64798 (Claim 33; Page 85-87);    JP05003790 (FIG. 6-8); WO99/46284 (FIG. 9); MIM:179780.

(20) IL20R-alpha (IL20Ra, ZCYTOR7)

Nucleotide

Genbank accession no AF184971

Genbank version no. AF184971.1 GI:6013324 (SEQ ID NO: 83)

Genbank record update date: Mar. 10, 2010 10:00 PM

Polypeptide

Genbank accession no. AAF01320

Genbank version no. AAF01320.1 GI:6013325 (SEQ ID NO: 84)

Genbank record update date: Mar. 10, 2010 10:00 PM

CROSS REFERENCES

-   Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J.,    et al Nature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19,    2001; Dumoutier L., et al J. Immunol. 167, 3545-3549,-   2001; Parrish-Novak J., et al J. Biol. Chem. 277, 47517-47523, 2002;    Pletnev S., et al (2003) 10 Biochemistry 42:12617-12624; Sheikh F.,    et al (2004) J. Immunol. 172, 2006-2010; EP1394274 (Example 11);    US2004/005320 (Example 5); WO2003/029262 (Page 74-75); WO2003/002717    (Claim 2; Page 63); WO2002/22153 (Page 45-47); US2002/042366 (Page    20-21); WO2001/46261 (Page 57-59); WO2001/46232 (Page 63-65);    WO98/37193 (Claim 1; Page 55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8;    EMBL; AF184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB)

Nucleotide

Genbank accession no AF229053

Genbank version no. AF229053.1 GI:10798902 (SEQ ID NO: 85)

Genbank record update date: Mar. 11, 2010 12:58 AM

Polypeptide

Genbank accession no. AAG23135

Genbank version no. AAG23135.1 GI:10798903 (SEQ ID NO: 86)

Genbank record update date: Mar. 11, 2010 12:58 AM

CROSS REFERENCES

-   Gary S. C., et al Gene 256, 139-147, 2000; Clark H. F., et al Genome    Res. 13, 2265-2270, 2003; Strausberg R. L., et al Proc. Natl. Acad.    Sci. U.S.A. 99, 16899-16903, 2002; US2003/186372 (Claim 11);    US2003/186373 (Claim 11); US2003/119131 (Claim 1; FIG. 52);    US2003/119122 (Claim 1; 20 FIG. 52); US2003/119126 (Claim 1);    US2003/119121 (Claim 1; FIG. 52); US2003/119129 (Claim 1);    US2003/119130 (Claim 1); US2003/119128 (Claim 1; FIG. 52);    US2003/119125 (Claim 1); WO2003/016475 (Claim 1); WO2002/02634    (Claim 1)

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5)

Nucleotide

Genbank accession no NM_004442

Genbank version no. NM_004442.6 GI:111118979 (SEQ ID NO: 87)

Genbank record update date: Sep. 8, 2012 04:43 PM

Polypeptide

Genbank accession no. NP_004433

Genbank version no. NP_004433.2 GI:21396504 (SEQ ID NO: 88)

Genbank record update date: Sep. 8, 2012 04:43 PM

CROSS REFERENCE

-   Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991) Oncogene    10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int.    Rev. Cytol. 196:177-244 (2000)); WO2003042661 (Claim 12);    WO200053216 (Claim 1; Page 41); WO2004065576 (Claim 1); WO2004020583    (Claim 9); WO2003004529 (Page 128-132); WO200053216 (Claim 1; Page    42); MIM:600997.

(23) ASLG659 (B7h)

Nucleotide

Genbank accession no. AX092328

Genbank version no. AX092328.1 GI:13444478 (SEQ ID NO: 89)

Genbank record update date: Jan. 26, 2011 07:37 AM

CROSS REFERENCES

-   US2004/0101899 (Claim 2); WO2003104399 (Claim 11); WO2004000221    (FIG. 3); US2003/165504 (Claim 1); US2003/124140 (Example 2);    US2003/065143 (FIG. 60); WO2002/102235 (Claim 13; Page 299);    US2003/091580 (Example 2); WO2002/10187 (Claim 6; FIG. 10);    WO2001/94641 (Claim 12; FIG. 7b); WO2002/02624 (Claim 13; FIG.    1A-1B); US2002/034749 (Claim 54; Page 45-46); WO2002/06317 (Example    2; Page 320-321, Claim 34; Page 321-322); WO2002/71928 (Page    468-469); WO2002/02587 (Example 1; FIG. 1); WO2001/40269 (Example 3;    Pages 190-192); WO2000/36107 (Example 2; Page 205-207);    WO2004/053079 (Claim 12); WO2003/004989 (Claim 1); WO2002/71928    (Page 233-234, 452-453); WO 01/16318.

(24) PSCA (Prostate stem cell antigen precursor)

Nucleotide

Genbank accession no AJ297436

Genbank version no. AJ297436.1 GI:9367211 (SEQ ID NO: 90)

Genbank record update date: Feb. 1, 2011 11:25 AM

Polypeptide

Genbank accession no. CAB97347

Genbank version no. CAB97347.1 GI:9367212 (SEQ ID NO: 91)

Genbank record update date: Feb. 1, 2011 11:25 AM

-   Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740,    1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem. Biophys.    Res. Commun. (2000) 275(3):783-788; WO2004/022709; EP1394274    (Example 11); US2004/018553 (Claim 17); WO2003/008537 (Claim 1);    WO2002/81646 (Claim 1; Page 164); WO2003/003906 (Claim 10; Page    288); WO2001/40309 (Example 1; FIG. 17); US2001/055751 (Example 1;    FIG. 1 b); WO2000/32752 (Claim 18; FIG. 1); WO98/51805 (Claim 17;    Page 97); WO98/51824 (Claim 10; Page 94); WO98/40403 (Claim 2; FIG.    1B); Accession: 043653; EMBL; AF043498; AAC39607.1

(25) GEDA

Nucleotide

Genbank accession no AY260763

Genbank version no. AY260763.1 GI:30102448 (SEQ ID NO: 92)

Genbank record update date: Mar. 11, 2010 02:24 AM

Polypeptide

Genbank accession no. AAP14954

Genbank version no. AAP14954.1 GI:30102449 (SEQ ID NO: 93)

Genbank record update date: Mar. 11, 2010 02:24 AM

CROSS REFERENCES

-   AP14954 lipoma HMGIC fusion-partnerlike protein/pid=AAP14954.1-Homo    sapiens (human); WO2003/054152 (Claim 20); WO2003/000842 (Claim 1);    WO2003/023013 (Example 3, Claim 20); US2003/194704 (Claim 45);    GI:30102449;

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3)

Nucleotide

Genbank accession no AF116456

Genbank version no. AF116456.1 GI:4585274 (SEQ ID NO: 94)

Genbank record update date: Mar. 10, 2010 09:44 PM

Polypeptide

Genbank accession no. AAD25356

Genbank version no. AAD25356.1 GI:4585275 (SEQ ID NO: 95)

Genbank record update date: Mar. 10, 2010 09:44 PM

CROSS REFERENCE

-   BAFF receptor/pid=NP_443177.1-Homo sapiens: Thompson, J. S., et al    Science 293 (5537), 2108-2111 (2001); WO2004/058309; WO2004/011611;    WO2003/045422 (Example; Page 32-33); WO2003/014294 (Claim 35; FIG.    6B); WO2003/035846 (Claim 70; Page 615-616); WO2002/94852 (Col    136-137); WO2002/38766 25 (Claim 3; Page 133); WO2002/24909 (Example    3; FIG. 3); MIM:606269; NP_443177.1; NM_052945_1; AF132600

(27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8,SIGLEC-2, FLJ22814)

Nucleotide

Genbank accession no AK026467

Genbank version no. AK026467.1 GI:10439337 (SEQ ID NO: 96)

Genbank record update date: Sep. 11, 2006 11:24 PM

Polypeptide

Genbank accession no. BAB15489

Genbank version no. BAB15489.1 GI:10439338 (SEQ ID NO: 97)

Genbank record update date: Sep. 11, 2006 11:24 PM

CROSS REFERENCES

-   Wilson et al (1991) J. Exp. Med. 173:137-146; 30 WO2003/072036    (Claim 1; FIG. 1); IM:107266; NP_001762.1; NM_001771_1.

(27a) CD22 (CD22 molecule)

Nucleotide

Genbank accession no X52785

Genbank version no. X52785.1 GI:29778 (SEQ ID NO: 98)

Genbank record update date: Feb. 2, 2011 10:09 AM

Polypeptide

Genbank accession no. CAA36988

Genbank version no. CAA36988.1 GI:29779 (SEQ ID NO: 99)

Genbank record update date: Feb. 2, 2011 10:09 AM

CROSS REFERENCES

-   Stamenkovic I. et al., Nature 345 (6270), 74-77 (1990)??

Other Information

Official Symbol: CD22

Other Aliases: SIGLEC-2, SIGLEC2

Other Designations: B-cell receptor CD22; B-lymphocyte cell adhesionmolecule; BL-CAM; CD22 antigen; T-cell surface antigen Leu-14; sialicacid binding Ig-like lectin 2; sialic acid-binding Ig-like lectin 2

Antibodies

-   G5/44 (Inotuzumab): DiJoseph J F., et al Cancer Immunol Immunother.    2005 January; 54(1):11-24.-   Epratuzumab-Goldenberg D M., et al Expert Rev Anticancer Ther.    6(10): 1341-53, 2006.

(28) CD79a (CD79A, CD79alpha), immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M

35 molecules, transduces a signal involved in B-cell differentiation),pl: 4.84, MW: 25028 TM: 2

[P] Gene Chromosome: 19q13.2).

Nucleotide

Genbank accession no NM_001783

Genbank version no. NM_001783.3 GI:90193587 (SEQ ID NO: 100)

Genbank record update date: Jun. 26, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_001774

Genbank version no. NP_001774.1 GI:4502685 (SEQ ID NO: 101)

Genbank record update date: Jun. 26, 2012 01:48 PM

CROSS REFERENCES

WO2003/088808, US2003/0228319; WO2003/062401 (claim 9); US2002/150573(claim 4, pages 13-14); WO99/58658 (claim 13, FIG. 16); WO92/07574 (FIG.1); U.S. Pat. No. 5,644,033; Ha et al (1992) J. Immunol.148(5):1526-1531; Müller et al (1992) Eur. J. Immunol. 22:1621-1625;Hashimoto et al (1994) Immunogenetics 40(4):287-295; Preud'homme et al(1992) Clin. Exp. 5 Immunol. 90(1):141-146; Yu et al (1992) J. Immunol.148(2) 633-637; Sakaguchi et al (1988) EMBO J. 7(11):3457-3464

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a 10 role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa,pl: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3,

Nucleotide

Genbank accession no NM_001716

Genbank version no. NM_001716.4 GI:342307092 (SEQ ID NO: 102)

Genbank record update date: Sep. 30, 2012 01:49 PM

Polypeptide

Genbank accession no. NP_001707

Genbank version no. NP_001707.1 GI:4502415 (SEQ ID NO: 103)

Genbank record update date: Sep. 30, 2012 01:49 PM

CROSS REFERENCES

-   WO2004/040000; WO2004/015426; US2003/105292 (Example 2); U.S. Pat.    No. 6,555,339 (Example 2); WO2002/61087 (FIG. 1); WO2001/57188    (Claim 20, page 269); WO2001/72830 (pages 12-13); WO2000/22129    (Example 1, pages 152-153, 15 Example 2, pages 254-256); WO99/28468    (claim 1, page 38); U.S. Pat. No. 5,440,021 (Example 2, col 49-52);    WO94/28931 (pages 56-58); WO92/17497 (claim 7, FIG. 5); Dobner et    al (1992) Eur. J. Immunol. 22:2795-2799; Barella et al (1995)    Biochem. J. 309:773-779

(30) HLA-DOB (Beta subunit of MHC class II molecule (la antigen) thatbinds peptides and 20 presents them to CD4+T lymphocytes); 273 aa, pl:6.56, MW: 30820. TM: 1 [P] Gene Chromosome: 6p21.3)

Nucleotide

Genbank accession no NM_002120

Genbank version no. NM_002120.3 GI:118402587 (SEQ ID NO: 104)

Genbank record update date: Sep. 8, 2012 04:46 PM

Polypeptide

Genbank accession no. NP_002111

Genbank version no. NP_002111.1 GI:4504403 (SEQ ID NO: 105)

Genbank record update date: Sep. 8, 2012 04:46 PM

CROSS REFERENCES

-   Tonnelle et al (1985) EMBO J. 4(11):2839-2847; Jonsson et al (1989)    Immunogenetics 29(6):411-413; Beck et al (1992) J. Mol. Biol.    228:433-441; Strausberg et al (2002) Proc. P Natl. Acad. Sci USA    99:16899-16903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766;    Beck et al (1996) J. Mol. Biol. 25 255:1-13; Naruse et al (2002)    Tissue Antigens 59:512-519; WO99/58658 (claim 13, FIG. 15); U.S.    Pat. No. 6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col    168-170); U.S. Pat. No. 6,011,146 (col 145-146); Kasahara et    al (1989) Immunogenetics 30(1):66-68; Larhammar et al (1985) J.    Biol. Chem. 260(26):14111-14119

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability); 422 aa), pl: 7.63,MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3).

Nucleotide

Genbank accession no NM_002561

Genbank version no. NM_002561.3 GI:325197202 (SEQ ID NO: 106)

Genbank record update date: Jun. 27, 2012 12:41 AM

Polypeptide

Genbank accession no. NP_002552

Genbank version no. NP_002552.2 GI:28416933 (SEQ ID NO: 107)

Genbank record update date: Jun. 27, 2012 12:41 AM

CROSS REFERENCES

-   Le et al (1997) FEBS Lett. 418(1-2):195-199; WO2004/047749;    WO2003/072035 (claim 10); Touchman et al (2000) Genome Res.    10:165-173; WO2002/22660 (claim 20); WO2003/093444 (claim 1);    WO2003/087768 (claim 1); WO2003/029277 (page 82)

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2); 359 aa, pl:8.66, MW: 40225, TM: 1 5 [P] Gene Chromosome: 9p13.3).

Nucleotide

Genbank accession no NM_001782

Genbank version no. NM_001782.2 GI:194018444 (SEQ ID NO: 108)

Genbank record update date: Jun. 26, 2012 01:43 PM

Polypeptide

Genbank accession no. NP_001773

Genbank version no. NP_001773.1 GI:4502683 (SEQ ID NO: 109)

Genbank record update date: Jun. 26, 2012 01:43 PM

CROSS REFERENCES

-   WO2004042346 (claim 65); WO2003/026493 (pages 51-52, 57-58);    WO2000/75655 (pages 105-106); Von Hoegen et al (1990) J. Immunol.    144(12):4870-4877; Strausberg et al (2002) Proc. Natl. Acad. Sci USA    99:16899-16903.

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis); 661 aa, pl:6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12).

Nucleotide

Genbank accession no NM_005582

Genbank version no. NM_005582.2 GI:167555126 (SEQ ID NO: 110)

Genbank record update date: Sep. 2, 2012 01:50 PM

Polypeptide

Genbank accession no. NP_005573

Genbank version no. NP_005573.2 GI:167555127 (SEQ ID NO: 111)

Genbank record update date: Sep. 2, 2012 01:50 PM

CROSS REFERENCES

-   US2002/193567; WO97/07198 (claim 11, pages 39-42); Miura et    al (1996) 15 Genomics 38(3):299-304; Miura et al (1998) Blood    92:2815-2822; WO2003/083047; WO97/44452 (claim 8, pages 57-61);    WO2000/12130 (pages 24-26).

(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte 20 differentiation); 429 aa, pl: 5.28,MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22)

Nucleotide

Genbank accession no NM_052938

Genbank version no. NM_052938.4 GI:226958543 (SEQ ID NO: 112)

Genbank record update date: Sep. 2, 2012 01:43 PM

Polypeptide

Genbank accession no. NP_443170

Genbank version no. NP_443170.1 GI:16418419 (SEQ ID NO: 113)

Genbank record update date: Sep. 2, 2012 01:43 PM

CROSS REFERENCES

-   WO2003/077836; WO2001/38490 (claim 6, FIGS. 18E-1-18-E-2); Davis et    al (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003/089624    (claim 8); EP1347046 (claim 1); WO2003/089624 (claim 7).

(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated2, a putative immunoreceptor with possible roles in B cell developmentand lymphomagenesis; deregulation of the gene by translocation occurs insome B cell malignancies); 977 aa, pl: 6.88, MW: 106468, TM: 1 [P] GeneChromosome: 1q21)

Nucleotide

Genbank accession no AF343662

Genbank version no. AF343662.1 GI:13591709 (SEQ ID NO: 114)

Genbank record update date: Mar. 11, 2010 01:16 AM

Polypeptide

Genbank accession no. AAK31325

Genbank version no. AAK31325.1 GI:13591710 (SEQ ID NO: 115)

Genbank record update date: Mar. 11, 2010 01:16 AM

CROSS REFERENCES

-   AF343663, AF343664, AF343665, AF369794, AF397453, AK090423,    AK090475, AL834187, AY358085; Mouse:AK089756, AY158090, AY506558;    NP_112571.1; WO2003/024392 (claim 2, FIG. 97); Nakayama et al (2000)    Biochem. Biophys. Res. Commun. 277(1):124-127; WO2003/077836;    WO2001/38490 (claim 3, FIGS. 18B-1-18B-2).

(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPPI, TR, putative transmembrane35 proteoglycan, related to the EGF/heregulin family of growth factorsand follistatin); 374 aa)

Nucleotide

Genbank accession no AF179274

Genbank version no. AF179274.2 GI:12280939 (SEQ ID NO: 116)

Genbank record update date: Mar. 11, 2010 01:05 AM

Polypeptide

Genbank accession no. AAD55776

Genbank version no. AAD55776.2 GI:12280940 (SEQ ID NO: 117)

Genbank record update date: Mar. 11, 2010 01:05 AM

CROSS REFERENCES

-   NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq:    NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5;    AY358907, CAF85723, CQ782436; WO2004/074320; JP2004113151;    WO2003/042661; WO2003/009814; EP1295944 (pages 69-70); WO2002/30268    (page 329); WO2001/90304; US2004/249130; US2004/022727;    WO2004/063355; US2004/197325; US2003/232350; 5 US2004/005563;    US2003/124579; Horie et al (2000) Genomics 67:146-152; Uchida et    al (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang et    al (2000) Cancer Res. 60:4907-12; Glynne-Jones et al (2001) Int J    Cancer. October 15; 94(2):178-84.

(37) PSMA—FOLH1 (Folate hydrolase (prostate-specific membrane antigen)1)

Nucleotide

Genbank accession no M99487

Genbank version no. M99487.1 GI:190663 (SEQ ID NO: 118)

Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA60209

Genbank version no. AAA60209.1 GI:190664 (SEQ ID NO: 119)

Genbank record update date: Jun. 23, 2010 08:48 AM

CROSS REFERENCES

-   Israeli R. S., et al Cancer Res. 53 (2), 227-230 (1993)

Other information

Official Symbol: FOLH1

Other Aliases: GIG27, FGCP, FOLH, GCP2, GCPII, NAALAD1, NAALAdase, PSM,PSMA, mGCP

Other Designations: N-acetylated alpha-linked acidic dipeptidase 1;N-acetylated-alpha-linked acidic dipeptidase I; NAALADase I; cellgrowth-inhibiting gene 27 protein; folylpoly-gamma-glutamatecarboxypeptidase; glutamate carboxylase II; glutamate carboxypeptidase2; glutamate carboxypeptidase II; membrane glutamate carboxypeptidase;prostate specific membrane antigen variant F;pteroylpoly-gamma-glutamate carboxypeptidase

Antibodies

U.S. Pat. No. 7,666,425:

Antibodies produces by Hybridomas having the following ATCCreferences:ATCC accession No. HB-12101, ATCC accession No. HB-12109,ATCC accession No. HB-12127 and ATCC accession No. HB-12126.

Proscan: a monoclonal antibody selected from the group consisting of8H12, 3E11, 17G1, 29B4, 30C1 and 20F2 (U.S. Pat. No. 7,811,564; MoffettS., et al Hybridoma (Larchmt). 2007 December; 26(6):363-72).

Cytogen: monoclonal antibodies 7E11-C5 (ATCC accession No. HB 10494) and9H10-A4 (ATCC accession No. HB11430)—U.S. Pat. No. 5,763,202

GlycoMimetics: NUH2-ATCC accession No. HB 9762 (U.S. Pat. No. 7,135,301)

Human Genome Science: HPRAJ70-ATCC accession No. 97131 (U.S. Pat. No.6,824,993); Amino acid sequence encoded by the cDNA clone (HPRAJ70)deposited as American Type Culture Collection (“ATCC”) Deposit No. 97131

Medarex: Anti-PSMA antibodies that lack fucosyl residues—U.S. Pat. No.7,875,278

Mouse anti-PSMA antibodies include the 3F5.4G6, 3D7.1.1, 4E10-1.14,3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6, 4C8B9,and monoclonal antibodies. Hybridomas secreting 3F5.4G6, 3D7.1.1,4E10-1.14, 3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6or 4C8B9 have been publicly deposited and are described in U.S. Pat. No.6,159,508. Relevant hybridomas have been publicly deposited and aredescribed in U.S. Pat. No. 6,107,090. Moreover, humanized anti-PSMAantibodies, including a humanized version of J591, are described infurther detail in PCT Publication WO 02/098897.

Other mouse anti-human PSMA antibodies have been described in the art,such as mAb 107-1A4 (Wang, S. et al. (2001) Int. J. Cancer 92:871-876)and mAb 2C9 (Kato, K. et al. (2003) Int. J. Urol. 10:439-444).

Examples of human anti-PSMA monoclonal antibodies include the 4A3, 7F12,8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 antibodies, isolated andstructurally characterized as originally described in PCT PublicationsWO 01/09192 and WO 03/064606 and in U.S. Provisional Application Ser.No. 60/654,125, entitled “Human Monoclonal Antibodies to ProstateSpecific Membrane Antigen (PSMA)”, filed on Feb. 18, 2005. The V.sub.Hamino acid sequences of 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and1C3 are shown in SEQ ID NOs: 1-9, respectively. The V.sub.L amino acidsequences of 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 areshown in SEQ ID NOs: 10-18, respectively.

Other human anti-PSMA antibodies include the antibodies disclosed in PCTPublication WO 03/034903 and US Application No. 2004/0033229.

NW Biotherapeutics: A hybridoma cell line selected from the groupconsisting of 3F5.4G6 having ATCC accession number HB12060, 3D7-1.I.having ATCC accession number HB12309, 4E10-1.14 having ATCC accessionnumber HB12310, 3E11 (ATCC HB12488), 4D8 (ATCC HB12487), 3E6 (ATCCHB12486), 3C9 (ATCC HB12484), 2C7 (ATCC HB12490), 1G3 (ATCC HB12489),3C4 (ATCC HB12494), 3C6 (ATCC HB12491), 4D4 (ATCC HB12493), 1G9 (ATCCHB12495), 5C8B9 (ATCC HB12492) and 3G6 (ATCC HB12485)—see U.S. Pat. No.6,150,508

PSMA Development Company/Progenics/Cytogen—Seattle Genetics: mAb 3.9,produced by the hybridoma deposited under ATCC Accession No. PTA-3258 ormAb 10.3, produced by the hybridoma deposited under ATCC Accession No.PTA-3347—U.S. Pat. No. 7,850,971

PSMA Development Company-Compositions of PSMA antibodies (US20080286284, Table 1)

-   This application is a divisional of U.S. patent application Ser. No.    10/395,894, filed on Mar. 21, 2003 (U.S. Pat. No. 7,850,971)-   University Hospital Freiburg, Germany—mAbs 3/A12, 3/E7, and 3/F11    (Wolf P., et al Prostate. 2010 Apr. 1; 70(5):562-9).

(38) SST (Somatostatin Receptor; note that there are 5 subtypes)

(38.1) SSTR2 (Somatostatin receptor 2)

Nucleotide

Genbank accession no NM_001050

Genbank version no. NM_001050.2 GI:44890054 (SEQ ID NO: 120)

Genbank record update date: Aug. 19, 2012 01:37 PM

Polypeptide

Genbank accession no. NP_001041

Genbank version no. NP_001041.1 GI:4557859 (SEQ ID NO: 121)

Genbank record update date: Aug. 19, 2012 01:37 PM

CROSS REFERENCES

-   Yamada Y., et al Proc. Natl. Acad. Sci. U.S.A. 89 (1), 251-255    (1992); Susini C., et al Ann Oncol. 2006 December; 17(12):1733-42

Other Information

Official Symbol: SSTR2

Other Designations: SRIF-1; SS2R; somatostatin receptor type 2

(38.2) SSTR5 (Somatostatin receptor 5)

Nucleotide

Genbank accession no D16827

Genbank version no. D16827.1 GI:487683 (SEQ ID NO: 122)

Genbank record update date: Aug. 1, 2006 12:45 PM

Polypeptide

Genbank accession no. BAAO4107

Genbank version no. BAA04107.1 GI:487684 (SEQ ID NO: 123)

Genbank record update date: Aug. 1, 2006 12:45 PM

CROSS REFERENCE

-   Yamada, Y., et al Biochem. Biophys. Res. Commun. 195 (2), 844-852    (1993)

Other Information

Official Symbol: SSTR5

Other Aliases: SS-5-R

Other Designations: Somatostatin receptor subtype 5; somatostatinreceptor type 5

(38.3) SSTR1

(38.4) SSTR3

(38.5) SSTR4

AvB6—Both subunits (39+40)

(39) ITGAV (Integrin, alpha V;

Nucleotide

Genbank accession no M14648 J02826 M18365

Genbank version no. M14648.1 GI:340306 (SEQ ID NO: 124)

Genbank record update date: Jun. 23, 2010 08:56 AM

Polypeptide

Genbank accession no. AAA36808

Genbank version no. AAA36808.1 GI:340307 (SEQ ID NO: 125)

Genbank record update date: Jun. 23, 2010 08:56 AM

CROSS REFERENCES

-   Suzuki S., et al Proc. Natl. Acad. Sci. U.S.A. 83 (22), 8614-8618    (1986)

Other information

Official Symbol: ITGAV

Other Aliases: CD51, MSK8, VNRA, VTNR

Other Designations: antigen identified by monoclonal antibody L230;integrin alpha-V; integrin alphaVbeta3; integrin, alpha V (vitronectinreceptor, alpha polypeptide, antigen CD51); vitronectin receptor subunitalpha

(40) ITGB6 (Integrin, beta 6)

Nucleotide

Genbank accession no NM_000888

Genbank version no. NM_000888.3 GI:9966771 (SEQ ID NO: 126)

Genbank record update date: Jun. 27, 2012 12:46 AM

Polypeptide

Genbank accession no. NP_000879

Genbank version no. NP_000879.2 GI:9625002 (SEQ ID NO: 127)

Genbank record update date: Jun. 27, 2012 12:46 AM

CROSS REFERENCES

-   Sheppard D. J., et al Biol. Chem. 265 (20), 11502-11507 (1990)

Other information

Official Symbol: ITGB6

Other Designations: integrin beta-6

Antibodies

Biogen: U.S. Pat. No. 7,943,742—Hybridoma clones 6.3G9 and 6.8G6 weredeposited with the ATCC, accession numbers ATCC PTA-3649 and -3645,respectively.

Biogen: U.S. Pat. No. 7,465,449—In some embodiments, the antibodycomprises the same heavy and light chain polypeptide sequences as anantibody produced by hybridoma 6.1A8, 6.3G9, 6.8G6, 6.2B1, 6.2B10,6.2A1, 6.2E5, 7.1G 10, 7.7G5, or 7.1C5.

Centocor (J&J): U.S. Pat. Nos. 7,550,142; 7,163,681

-   -   For example in U.S. Pat. No. 7,550,142—an antibody having human        heavy chain and human light chain variable regions comprising        the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8.

Seattle Genetics: 15H3 (Ryan M C., et al Cancer Res Apr. 15, 2012; 72(8Supplement): 4630)

(41) CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5)

Nucleotide

Genbank accession no M17303

Genbank version no. M17303.1 GI:178676 (SEQ ID NO: 128)

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAB59513

Genbank version no. AAB59513.1 GI:178677 (SEQ ID NO: 129)

Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS REFERENCES

-   Beauchemin N., et al Mol. Cell. Biol. 7 (9), 3221-3230 (1987)

Other information

Official Symbol: CEACAM5

Other Aliases: CD66e, CEA

Other Designations: meconium antigen 100

Antibodies

AstraZeneca-Medlmmune:US 20100330103; US20080057063;

-   -   US20020142359        -   for example an antibody having complementarity determining            regions (CDRs) with the following sequences: heavy chain;            CDR1—DNYMH (SEQ ID NO: 216), CDR2—WIDPENGDTE YAPKFRG (SEQ ID            NO: 217), CDR3-LIYAGYLAMD Y (SEQ ID NO: 218); and light            chain CDR1—SASSSVTYMH (SEQ ID NO: 219), CDR2—STSNLAS (SEQ ID            NO: 220), CDR3-QQRSTYPLT (SEQ ID NO: 221).    -   Hybridoma 806.077 deposited as European Collection of Cell        Cultures (ECACC) deposit no. 96022936.

Research Corporation Technologies, Inc.:U.S. Pat. No. 5,047,507

Bayer Corporation: U.S. Pat. No. 6,013,772

BioAlliance: U.S. Pat. Nos. 7,982,017; 7,674,605

-   -   U.S. Pat. No. 7,674,605        -   an antibody comprising the heavy chain variable region            sequence from the amino acid sequence of SEQ ID NO: 1, and            the light chain variable region sequence from the amino acid            sequence of SEQ ID NO:2.        -   an antibody comprising the heavy chain variable region            sequence from the amino acid sequence of SEQ ID NO:5, and            the light chain variable region sequence from the amino acid            sequence of SEQ ID NO:6.

Celltech Therapeutics Limited: U.S. Pat. No. 5,877,293

The Dow Chemical Company: U.S. Pat. Nos. 5,472,693; 6,417,337; 6,333,405

-   -   U.S. Pat. No. 5,472,693—for example, ATCC No. CRL-11215    -   U.S. Pat. No. 6,417,337—for example, ATCC CRL-12208    -   U.S. Pat. No. 6,333,405—for example, ATCC CRL-12208

Immunomedics, Inc: U.S. Pat. Nos. 7,534,431; 7,230,084; 7,300,644;6,730,300;

-   -   US20110189085        -   an antibody having CDRs of the light chain variable region            comprise: CDR1 comprises KASQDVGTSVA (SEQ ID NO: 20) (SEQ ID            NO: 222); CDR2 comprises WTSTRHT (SEQ ID NO: 21) (SEQ ID NO:            223); and CDR3 comprises QQYSLYRS (SEQ ID NO: 22) (SEQ ID            NO: 224);        -   and the CDRs of the heavy chain variable region of said            anti-CEA antibody comprise: CDR1 comprises TYWMS (SEQ ID            NO: 23) (SEQ ID NO: 225); CDR2 comprises EIHPDSSTINYAPSLKD            (SEQ ID NO: 24) (SEQ ID NO: 226) and CDR3 comprises            LYFGFPWFAY (SEQ ID NO: 25) (SEQ ID NO: 227).    -   US201 00221175; US20090092598; US20070202044; US20110064653;        US20090185974; US20080069775.

(42) MET (met proto-oncogene; hepatocyte growth factor receptor)

Nucleotide

Genbank accession no M35073

Genbank version no. M35073.1 GI:187553 (SEQ ID NO: 130)

Genbank record update date: Mar. 6, 2012 11:12 AM

Polypeptide

Genbank accession no. AAA59589

Genbank version no. AAA59589.1 GI:553531 (SEQ ID NO: 131)

Genbank record update date: Mar. 6, 2012 11:12 AM

CROSS REFERENCES

-   Dean M., et al Nature 318 (6044), 385-388 (1985)

Other information

Official Symbol: MET

Other Aliases: AUTS9, HGFR, RCCP2, c-Met

Other Designations: HGF receptor; HGF/SF receptor; SF receptor;hepatocyte growth factor receptor; met proto-oncogene tyrosine kinase;proto-oncogene c-Met; scatter factor receptor; tyrosine-protein kinaseMet

Antibodies

Abgenix/Pfizer: US20100040629

-   -   for example, the antibody produced by hybridoma 13.3.2 having        American Type Culture Collection (ATCC) accession number        PTA-5026; the antibody produced by hybridoma 9.1.2 having ATCC        accession number PTA-5027; the antibody produced by hybridoma        8.70.2 having ATCC accession number PTA-5028; or the antibody        produced by hybridoma 6.90.3 having ATCC accession number        PTA-5029.

Amgen/Pfizer: US20050054019

-   -   for example, an antibody comprising a heavy chain having the        amino acid sequences set forth in SEQ ID NO: 2 where X2 is        glutamate and X4 is serine and a light chain having the amino        acid sequence set forth in SEQ ID NO: 4 where X8 is alanine,        without the signal sequences; an antibody comprising a heavy        chain having the amino acid sequences set forth in SEQ ID NO: 6        and a light chain having the amino acid sequence set forth in        SEQ ID NO: 8, without the signal sequences; an antibody        comprising a heavy chain having the amino acid sequences set        forth in SEQ ID NO: 10 and a light chain having the amino acid        sequence set forth in SEQ ID NO: 12, without the signal        sequences; or an antibody comprising a heavy chain having the        amino acid sequences set forth in SEQ ID NO: 14 and a light        chain having the amino acid sequence set forth in SEQ ID NO: 16,        without the signal sequences.

Agouron Pharmaceuticals (Now Pfizer): US20060035907

Eli Lilly: US20100129369

Genentech: U.S. Pat. No. 5,686,292; US20100028337; US20100016241;US20070129301; US 20070098707; US20070092520, US20060270594;US20060134104; US20060035278; US 20050233960; US20050037431

-   -   U.S. Pat. No. 5,686,292—for example, ATCC HB-11894 and ATCC        HB-11895    -   US 20100016241—for example, ATCC HB-11894 (hybridoma 1A3.3.13)        or HB-11895 (hybridoma 5D5.11.6)

National Defense Medical Center, Taiwan: Lu R M., et al Biomaterials.2011 April; 32(12):3265-74.

Novartis: US20090175860

-   -   for example, an antibody comprising the sequences of CDR1, CDR2        and CDR3 of heavy chain 4687, wherein the sequences of CDR1,        CDR2, and CDR3 of heavy chain 4687 are residues 26-35, 50-65,        and 98-102, respectively, of SEQ ID NO: 58; and the sequences of        CDR1, CDR2, and CDR3 of light chain 5097, wherein the sequences        of CDR1, CDR2, and CDR3 of light chain 5097 are residues        24-39,55-61, and 94-100 of SEQ ID NO: 37.

Pharmacia Corporation: US20040166544

Pierre Fabre: US20110239316, US20110097262, US20100115639

Sumsung: US 20110129481—for example a monoclonal antibody produced froma hybridoma cell having accession number KCLRF-BP-00219 or accessionnumber of KCLRF-BP-00223.

Samsung: US 20110104176—for example an antibody produced by a hybridomacell having Accession Number: KCLRF-BP-00220.

University of Turin Medical School: DN-30 Pacchiana G., et al J BiolChem. 2010 Nov. 12; 285(46):36149-57

Van Andel Research Institute: Jiao Y., et al Mol Biotechnol. 2005September; 31(1):41-54.

(43) MUC1 (Mucin 1, cell surface associated)

Nucleotide

Genbank accession no J05581

Genbank version no. J05581.1 GI:188869 (SEQ ID NO: 132)

Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA59876

Genbank version no. AAA59876.1 GI:188870 (SEQ ID NO: 133)

Genbank record update date: Jun. 23, 2010 08:48 AM

CROSS REFERENCES

-   Gendler S. J., et al J. Biol. Chem. 265 (25), 15286-15293 (1990)

Other information

Official Symbol: MUC1

Other Aliases: RP11-263K19.2, CD227, EMA, H23AG, KL-6, MAM6, MUC-1,MUC-1/SEC,

MUC-1/X, MUC1/ZD, PEM, PEMT, PUM

Other Designations: DF3 antigen; H23 antigen; breastcarcinoma-associated antigen DF3; carcinoma-associated mucin; episialin;krebs von den Lungen-6; mucin 1, transmembrane; mucin-1; peanut-reactiveurinary mucin; polymorphic epithelial mucin; tumor associated epithelialmucin; tumor-associated epithelial membrane antigen; tumor-associatedmucin

Antibodies

AltaRex-Quest Pharma Tech: U.S. Pat. No. 6,716,966—for example an Alt-1antibody produced by the hybridoma ATCC No PTA-975.

AltaRex-Quest Pharma Tech: U.S. Pat. No. 7,147,850

-   CRT: 5E5—Sorensen AL., et al Glycobiology vol. 16 no. 2 pp. 96-107,    2006; HMFG2—Burchell J., et al Cancer Res., 47, 5476-5482 (1987)

Glycotope GT-MAB: GT-MAB 2.5-GEX (Website:

http://www.glycotope.com/pipeline/pankomab-gex)

Immunogen: U.S. Pat. No. 7,202,346

-   -   for example, antibody MJ-170: hybridoma cell line MJ-170 ATCC        accession no. PTA-5286Monoclonal antibody MJ-171: hybridoma cell        line MJ-171 ATCC accession no. PTA-5287; monoclonal antibody        MJ-172: hybridoma cell line MJ-172 ATCC accession no. PTA-5288;        or monoclonal antibody MJ-173: hybridoma cell line MJ-173 ATCC        accession no. PTA-5302

Immunomedics: U.S. Pat. No. 6,653,104

Ramot Tel Aviv Uni: U.S. Pat. No. 7,897,351

Regents Uni. CA: U.S. Pat. No. 7,183,388; US20040005647; US20030077676.

Roche GlycArt: U.S. Pat. No. 8,021,856

Russian National Cancer Research Center: Imuteran-Ivanov P K., et alBiotechnol J. 2007 July; 2(7):863-70

Technische Univ Braunschweig: (llB6, HT186-B7, HT186-D11, HT186-G2,HT200-3A-C1, HT220-M-D1, HT220-M-G8)—Thie H., et al PLoS One. 2011 Jan.14; 6(1):e15921

(44) CA9 (Carbonic anhydrase IX)

Nucleotide

Genbank accession no. X66839

Genbank version no. X66839.1 GI:1000701 (SEQ ID NO: 134)

Genbank record update date: Feb. 2, 2011 10:15 AM

Polypeptide

Genbank accession no. CAA47315

Genbank version no. CAA47315.1 GI:1000702 (SEQ ID NO: 135)

Genbank record update date: Feb. 2, 2011 10:15 AM

CROSS REFERENCE

-   Pastorek J., et al Oncogene 9 (10), 2877-2888 (1994)

Other information

Official Symbol: CA9

Other Aliases: CAIX, MN

Other Designations: CA-IX; P54/58N; RCC-associated antigen G250;RCC-associated protein G250; carbonate dehydratase IX; carbonicanhydrase 9; carbonic dehydratase; membrane antigen MN; pMW1; renal cellcarcinoma-associated antigen G250

Antibodies

Abgenix/Amgen: US20040018198

Affibody: Anti-CAIX Affibody molecules

-   -   (http://www.affibody.com/en/Product-Portfolio/Pipeline/)

Bayer: U.S. Pat. No. 7,462,696

Bayer/Morphosys: 3ee9 mAb—Petrul H M., et al Mol Cancer Ther. 2012February; 11(2):340-9

Harvard Medical School: Antibodies G10, G36, G37, G39, G45, G57, G106,G119, G6, G27, G40 and G125. Xu C., et al PLoS One. 2010 Mar. 10;5(3):e9625

Institute of Virology, Slovak Academy of Sciences (Bayer)—U.S. Pat. No.5,955,075

-   -   for example, M75-ATCC Accession No. HB 11128 or MN12—ATCC        Accession No. HB 11647

Institute of Virology, Slovak Academy of Sciences: U.S. Pat. No.7,816,493

-   -   for example the M75 monoclonal antibody that is secreted from        the hybridoma VU-M75, which was deposited at the American Type        Culture Collection under ATCC No. HB 11128; or the V/10        monoclonal antibody secreted from the hybridoma V/10-VU, which        was deposited at the International Depository Authority of the        Belgian Coordinated Collection of Microorganisms (BCCM) at the        Laboratorium voor Moleculaire Bioloqie-Plasmidencollectie (LMBP)        at the Universeit Gent in Gent, Belgium, under Accession No.        LMBP 6009CB.

Institute of Virology, Slovak Academy of Sciences US20080177046;US20080176310; US 20080176258; US20050031623

Novartis: US20090252738

Wilex: U.S. Pat. No. 7,691,375—for example the antibody produced by thehybridoma cell line DSM ASC 2526.

Wilex: US20110123537; Rencarex: Kennett R H., et al Curr Opin Mol Ther.2003 February; 5(1):70-5

Xencor: US20090162382

(45) EGFRvIII (Epidermal growth factor receptor (EGFR), transcriptvariant 3,

Nucleotide

Genbank accession no. NM_201283

Genbank version no. NM_201283.1 GI:41327733 (SEQ ID NO: 136)

Genbank record update date: Sep. 30, 2012 01:47 PM

Polypeptide

Genbank accession no. NP_958440

Genbank version no. NP_958440.1 GI:41327734 (SEQ ID NO: 137)

Genbank record update date: Sep. 30, 2012 01:47 PM

CROSS-REFERENCES

-   Batra S K., et al Cell Growth Differ 1995; 6:1251-1259.

Antibodies:

U.S. Pat. Nos. 7,628,986 and 7,736,644 (Amgen)

-   -   For example, a heavy chain variable region amino acid sequence        selected from the group consisting of SEQ ID NO: 142 and        variants & a light chain variable region amino acid sequence        selected from the group consisting of: SEQ ID NO: 144 and        variants.

US20100111979 (Amgen)

-   -   For example, an antibody comprising a heavy chain amino acid        sequence comprising:    -   CDR1 consisting of a sequence selected from the group consisting        of the amino acid sequences for the CDR1 region of antibodies        13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),        150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139        (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318        (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17);    -   CDR2 consisting of a sequence selected from the group consisting        of the amino acid sequences for the CDR2 region of antibodies        13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),        150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139        (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318        (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17);        and    -   CDR3 consisting of a sequence selected from the group consisting        of the amino acid sequences for the CDR3 region of antibodies        13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),        150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139        (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318        (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17).

US20090240038 (Amgen)

-   -   For example, an antibody having at least one of the heavy or        light chain polypeptides comprises an amino acid sequence that        is at least 90% identical to the amino acid sequence selected        from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ        ID NO: 142, SEQ ID NO: 144, and any combination thereof.

US20090175887 (Amgen)

-   -   For example, an antibody having a heavy chain amino acid        sequence selected from the group consisting of the heavy chain        amino acid sequence of antibody 13.1.2 (SEQ ID NO: 138), 131        (SEQ ID NO: 2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ        ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID        NO: 12), 124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID        NO: 16), and 333 (SEQ ID NO: 17).

US20090156790 (Amgen)

-   -   For example, antibody having heavy chain polypeptide and a light        chain polypeptide, wherein at least one of the heavy or light        chain polypeptides comprises an amino acid sequence that is at        least 90% identical to the amino acid sequence selected from the        group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO:        142, SEQ ID NO: 144, and any combination thereof.

US20090155282, US20050059087 and US20050053608 (Amgen)

For example, an antibody heavy chain amino acid sequence selected fromthe group consisting of the heavy chain amino acid sequence of antibody13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4), 150(SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO:10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342(SEQ ID NO: 16), and 333 (SEQ ID NO: 17).

MR1-1 (U.S. Pat. No. 7,129,332; Duke)

For example, a variant antibody having the sequence of SEQ ID NO.18 withthe substitutions S98P-T99Y in the CDR3 VH, and F92W in CDR3 VL.

L8A4, H10, Y10 (Wikstrand C J., et al Cancer Res. 1995 Jul. 15;55(14):3140-8; Duke)

US20090311803 (Harvard University)

-   -   For example, SEQ ID NO:9 for antibody heavy chain variable        region, and SEQ ID NO: 3 for light chain variable region amino        acid sequences

US20070274991 (EMD72000, also known as matuzumab; Harvard University)

-   -   For example, SEQ ID NOs: 3 & 9 for light chain and heavy chain        respectively

U.S. Pat. No. 6,129,915 (Schering)

-   -   For example, SEQ. ID NOs: 1, 2, 3, 4, 5 and 6.

mAb CH12—Wang H., et al FASEB J. 2012 January; 26(1):73-80 (ShanghaiCancer Institute).

RAbDMvIII—Gupta P., et al BMC Biotechnol. 2010 Oct. 7; 10:72 (StanfordUniversity Medical Center).

mAb Ua30—Ohman L., et al Tumour Biol. 2002 March-April; 23(2):61-9(Uppsala University).

Han D G., et al Nan Fang Yi Ke Da Xue Xue Bao. 2010 January; 30(1):25-9(Xi'an Jiaotong University).

(46) CD33 (CD33 molecule)

Nucleotide

Genbank accession no. M_23197

Genbank version no. NM_23197.1 GI:180097 (SEQ ID NO: 138)

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA51948

Genbank version no. AAA51948.1 GI:188098 (SEQ ID NO: 139)(**GI:180098)

Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS-REFERENCES

-   Simmons D., et al J. Immunol. 141 (8), 2797-2800 (1988)

Other information

Official Symbol: CD33

Other Aliases: SIGLEC-3, SIGLEC3, p67

Other Designations: CD33 antigen (gp67); gp67; myeloid cell surfaceantigen CD33; sialic acid binding Ig-like lectin 3; sialic acid-bindingIg-like lectin

Antibodies

-   H195 (Lintuzumab)-Raza A., et al Leuk Lymphoma. 2009 August;    50(8):1336-44; U.S. Pat. No. 6,759,045 (Seattle    Genetics/Immunomedics)-   mAb OKT9: Sutherland, D. R. et al. Proc Natl Acad Sci USA 78(7):    4515-4519 1981, Schneider, C., et al J Biol Chem 257, 8516-8522    (1982)-   mAb E6: Hoogenboom, H. R., et al J Immunol 144, 3211-3217 (1990)-   U.S. Pat. No. 6,590,088 (Human Genome Sciences)    -   For example, SEQ ID NOs: 1 and 2 and ATCC accession no. 97521-   U.S. Pat. No. 7,557,189 (Immunogen)    -   For example, an antibody or fragment thereof comprising a heavy        chain variable region which comprises three CDRs having the        amino acid sequences of SEQ ID NOs:1-3 and a light chain        variable region comprising three CDRs having the amino acid        sequences of SEQ ID NOs:4-6.

(47) CD19 (CD19 molecule)

Nucleotide

Genbank accession no. NM_001178098

Genbank version no. NM_001178098.1 GI:296010920 (SEQ ID NO: 140)

Genbank record update date: Sep. 10, 2012 12:43 AM

Polypeptide

Genbank accession no. NP_001171569

Genbank version no. NP_001171569.1 GI:296010921 (SEQ ID NO: 141)

Genbank record update date: Sep. 10, 2012 12:43 AM

CROSS-REFERENCES

-   Tedder T F., et al J. Immunol. 143 (2): 712-7 (1989)

Other information

Official Symbol: CD19

Other Aliases: B4, CVID3

Other Designations: B-lymphocyte antigen CD19; B-lymphocyte surfaceantigen B4; T-cell surface antigen Leu-12; differentiation antigen CD19

Antibodies

Immunogen: HuB4—Al-Katib A M., et al Clin Cancer Res. 2009 Jun. 15;15(12):4038-45.

4G7: Kügler M., et al Protein Eng Des Sel. 2009 March; 22(3):135-47

For example, sequences in FIG. 3 of of Knappik, A. et al. J Mol Biol2000 February; 296(1):57-86

AstraZeneca/MedImmune: MEDI-551—Herbst R., et al J Pharmacol Exp Ther.2010 October; 335(1):213-22

Glenmark Pharmaceuticals: GBR-401—Hou S., et al Mol Cancer Ther November2011 10 (Meeting Abstract Supplement) C164

U.S. Pat. No. 7,109,304 (Immunomedics)

-   -   For example, an antibody comprising the sequence of hA19Vk (SEQ        ID NO:7) and the sequence of hA19VH (SEQ ID NO:10)

U.S. Pat. No. 7,902,338 (Immunomedics)

-   -   For example, an antibody or antigen-binding fragment thereof        that comprises the light chain complementarity determining        region CDR sequences CDR1 of SEQ ID NO: 16 (KASQSVDYDGDSYLN)        (SEQ ID NO: 228); CDR2 of SEQ ID NO: 17 (DASNLVS) (SEQ ID NO:        229); and CDR3 of SEQ ID NO: 18 (QQSTEDPWT) (SEQ ID NO: 230) and        the heavy chain CDR sequences CDR1 of SEQ ID NO: 19 (SYWMN) (SEQ        ID NO: 231); CDR2 of SEQ ID NO: 20 (QIWPGDGDTNYNGKFKG) (SEQ ID        NO: 232) and CDR3 of SEQ ID NO: 21 (RETTTVGRYYYAMDY) (SEQ ID        NO: 233) and also comprises human antibody framework (FR) and        constant region sequences with one or more framework region        amino acid residues substituted from the corresponding framework        region sequences of the parent murine antibody, and wherein said        substituted FR residues comprise the substitution of serine for        phenylalanine at Kabat residue 91 of the heavy chain variable        region.

Medarex: MDX-1342—Cardarelli P M., et al Cancer Immunol Immunother. 2010February; 59(2):257-65.

MorphoSys/Xencor: MOR-208/XmAb-5574—Zalevsky J., et al Blood. 2009 Apr.16; 113(16):3735-43

U.S. Pat. No. 7,968,687 (Seattle Genetics)

-   -   An antibody or antigen-binding fragment comprising a heavy chain        variable domain comprising the amino acid sequence of SEQ ID        NO:9 and a light chain variable domain comprising the amino acid        sequence of SEQ ID NO: 24.

4G7 chim—Lang P., et al Blood. 2004 May 15; 103(10):3982-5 (Universityof Tübingen)

-   -   For example, FIG. 6 and SEQ ID No: 80 of US20120082664

Zhejiang University School of Medicine: 2E8—Zhang J., et al J DrugTarget. 2010 November; 18(9):675-8

(48) IL2RA (Interleukin 2 receptor, alpha); NCBI Reference Sequence:NM_000417.2);

Nucleotide

Genbank accession no. NM_000417

Genbank version no. NM_000417.2 GI:269973860 (SEQ ID NO: 142)

Genbank record update date: Sep. 9, 2012 04:59 PM

Polypeptide

Genbank accession no. NP_000408

Genbank version no. NP_000408.1 GI:4557667 (SEQ ID NO: 143)

Genbank record update date: Sep. 9, 2012 04:59 PM

CROSS-REFERENCES

-   Kuziel W. A., et al J. Invest. Dermatol. 94 (6 SUPPL), 27S-32S    (1990)

Other information

Official Symbol: IL2RA

Other Aliases: RP11-536K7.1, CD25, IDDM10, IL2R, TCGFR

Other Designations: FIL-2 receptor subunit alpha; IL-2-RA; IL-2R subunitalpha; IL2-RA;

TAC antigen; interleukin-2 receptor subunit alpha; p55

Antibodies

U.S. Pat. No. 6,383,487 (Novartis/UCL: Baxilisimab [Simulect])

U.S. Pat. No. 6,521,230 (Novartis/UCL: Baxilisimab [Simulect])

-   -   For example, an antibody having an antigen binding site        comprises at least one domain which comprises CDR1 having the        amino acid sequence in SEQ. ID. NO: 7, CDR2 having the amino        acid sequence in SEQ. ID. NO: 8, and CDR3 chaving the amino acid        sequence in SEQ. ID. NO: 9; or said CDR1, CDR2 and CDR3 taken in        sequence as a whole comprise an amino acid sequence which is at        least 90% identical to SEQ. ID. NOs: 7, 8 and 9 taken in        sequence as a whole.

Daclizumab—Rech A J., et al Ann N YAcad Sci. 2009 September; 1174:99-106(Roche)

(49) AXL (AXL receptor tyrosine kinase)

Nucleotide

Genbank accession no. M76125

Genbank version no. M76125.1 GI:292869 (SEQ ID NO: 144)

Genbank record update date: Jun. 23, 2010 08:53 AM

Polypeptide

Genbank accession no. AAA61243

Genbank version no. AAA61243.1 GI:29870 (SEQ ID NO: 145)(**GI:292870)

Genbank record update date: Jun. 23, 2010 08:53 AM

CROSS-REFERENCES

-   O'Bryan J. P., et al Mol. Cell. Biol. 11 (10), 5016-5031 (1991);    Bergsagel P. L., et al J. Immunol. 148 (2), 590-596 (1992)

Other information

Official Symbol: AXL

Other Aliases: JTK11, UFO

Other Designations: AXL oncogene; AXL transforming sequence/gene;oncogene AXL; tyrosine-protein kinase receptor UFO

Antibodies

YW327.6S2—Ye X., et al Oncogene. 2010 Sep. 23; 29(38):5254-64.(Genentech)

BergenBio: BGB324 (http://www.bergenbio.com/BGB324)

(50) CD30—TNFRSF8 (Tumor necrosis factor receptor superfamily, member 8)

Nucleotide

Genbank accession no. M83554

Genbank version no. M83554.1 GI:180095 (SEQ ID NO: 146)

Genbank record update date: Jun. 23, 2010 08:53 AM

Polypeptide

Genbank accession no. AAA51947

Genbank version no. AAA51947.1 GI:180096 (SEQ ID NO: 147)

Genbank record update date: Jun. 23, 2010 08:53 AM

CROSS REFERENCE

-   Durkop H., et al Cell 68 (3), 421-427 (1992)

Other Information

Official Symbol: TNFRSF8

Other Aliases: CD30, D1S166E, Ki-1

Other Designations: CD30L receptor; Ki-1 antigen; cytokine receptorCD30; lymphocyte activation antigen CD30; tumor necrosis factor receptorsuperfamily member 8

(51) BCMA (B-cell maturation antigen)—TNFRSF17 (Tumor necrosis factorreceptor superfamily, member 17)

Nucleotide

Genbank accession no. Z29574

Genbank version no. Z29574.1 GI:471244 (SEQ ID NO: 148)

Genbank record update date: Feb. 2, 2011 10:40 AM

Polypeptide

Genbank accession no. CAA82690

Genbank version no. CAA82690.1 GI:471245 (SEQ ID NO: 149)

Genbank record update date: Feb. 2, 2011 10:40 AM

CROSS-REFERENCES

-   Laabi Y., et al Nucleic Acids Res. 22 (7), 1147-1154 (1994)

Other Information

Official Symbol: TNFRSF17

Other Aliases: BCM, BCMA, CD269

Other Designations: B cell maturation antigen; B-cell maturation factor;B-cell maturation protein; tumor necrosis factor receptor superfamilymember 17

(52) CT Ags—CTA (Cancer Testis Antigens)

CROSS-REFERENCES

-   Fratta E., et al. Mol Oncol. 2011 April; 5(2):164-82; Lim S H., at    al Am J Blood Res. 2012; 2(1):29-35.

(53) CD174 (Lewis Y)—FUT3 (fucosyltransferase 3 (galactoside3(4)-L-fucosyltransferase, Lewis blood group)

Nucleotide

Genbank accession no. NM000149

Genbank version no. NM000149.3 GI:148277008 (SEQ ID NO: 150)

Genbank record update date: Jun. 26, 2012 04:49 PM

Polypeptide

Genbank accession no. NP_000140

Genbank version no. NP_000140.1 GI:4503809 (SEQ ID NO: 151)

Genbank record update date: Jun. 26, 2012 04:49 PM

CROSS-REFERENCES

-   Kukowska-Latallo, J. F., et al Genes Dev. 4 (8), 1288-1303 (1990)

Other Information

Official Symbol: FUT3

Other Aliases: CD174, FT3B, FucT-III, LE, Les

Other Designations: Lewis F T; alpha-(1,3/1,4)-fucosyltransferase; bloodgroup Lewis alpha-4-fucosyltransferase; fucosyltransferase III;galactoside 3(4)-L-fucosyltransferase

(54) CLECI4A (C-type lectin domain family 14, member A; Genbankaccession no. NM175060)

Nucleotide

Genbank accession no. NM175060

Genbank version no. NM175060.2 GI:371123930 (SEQ ID NO: 152)

Genbank record update date: Apr. 1, 2012 03:34 PM

Polypeptide

Genbank accession no. NP_778230

Genbank version no. NP_778230.1 GI:28269707 (SEQ ID NO: 153)

Genbank record update date: Apr. 1, 2012 03:34 PM

Other Information

Official Symbol: CLEC14A

Other Aliases: UNQ236/PRO269, C14orf27, CEG1, EGFR-5

Other Designations: C-type lectin domain family 14 member A; CIECT andEGF-like domain containing protein; epidermal growth factor receptor 5

(55) GRP78—HSPA5 (heat shock 70 kDa protein 5 (glucose-regulatedprotein, 78 kDa)

Nucleotide

Genbank accession no. NM005347

Genbank version no. NM005347.4 GI:305855105 (SEQ ID NO: 154)

Genbank record update date: Sep. 30, 2012 01:42 PM

Polypeptide

Genbank accession no. NP_005338

Genbank version no. NP_005338.1 GI:16507237 (SEQ ID NO: 155)

Genbank record update date: Sep. 30, 2012 01:42 PM

CROSS-REFERENCES

-   Ting J., et al DNA 7 (4), 275-286 (1988)

Other Information

Official Symbol: HSPA5

Other Aliases: BIP, GRP78, MIF2

Other Designations: 78 kDa glucose-regulated protein; endoplasmicreticulum lumenal Ca(2+)-binding protein grp78; immunoglobulin heavychain-binding protein

(56) CD70 (CD70 molecule) L08096

Nucleotide

Genbank accession no. L08096

Genbank version no. L08096.1 GI:307127 (SEQ ID NO: 156)

Genbank record update date: Jun. 23, 2012 08:54 AM

Polypeptide

Genbank accession no. AAA36175

Genbank version no. AAA36175.1 GI:307128 (SEQ ID NO: 157)

Genbank record update date: Jun. 23, 2012 08:54 AM

CROSS-REFERENCES

-   Goodwin R. G., et al Cell 73 (3), 447-456 (1993)

Other Information

Official Symbol: CD70

Other Aliases: CD27L, CD27LG, TNFSF7

Other Designations: CD27 ligand; CD27-L; CD70 antigen; Ki-24 antigen;surface antigen CD70; tumor necrosis factor (ligand) superfamily, member7; tumor necrosis factor ligand superfamily member 7

Antibodies

MDX-1411 against CD70 (Medarex)

h1F6 (Oflazoglu, E., et al, Clin Cancer Res. 2008 Oct. 1;14(19):6171-80; Seattle Genetics)

-   -   For example, see US20060083736 SEQ ID NOs: 1, 2, 11 and 12 and        FIG. 1.

(57) Stem Cell specific antigens. For example:

-   -   5T4 (see entry (63) below)    -   CD25 (see entry (48) above)    -   CD32        -   Polypeptide            -   Genbank accession no. ABK42161            -   Genbank version no. ABK42161.1 GI:117616286 (SEQ ID NO:                158)            -   Genbank record update date: Jul. 25, 2007 03:00 PM    -   LGR5/GPR49        -   Nucleotide            -   Genbank accession no. NM_003667            -   Genbank version no. NM_003667.2 GI:24475886 (SEQ ID NO:                159)            -   Genbank record update date: Jul. 22, 2012 03:38 PM        -   Polypeptide            -   Genbank accession no. NP_003658            -   Genbank version no. NP_003658.1 GI:4504379 (SEQ ID NO:                160)            -   Genbank record update date: Jul. 22, 2012 03:38 PM    -   Prominin/CD133        -   Nucleotide            -   Genbank accession no. NM_006017            -   Genbank version no. NM_006017.2 GI:224994187 (SEQ ID NO:                161)            -   Genbank record update date: Sep. 30, 2012 01:47 PM        -   Polypeptide            -   Genbank accession no. NP_006008            -   Genbank version no. NP_006008.1 GI:5174387 (SEQ ID NO:                162)            -   Genbank record update date: Sep. 30, 2012 01:47 PM

(58) ASG-5

CROSS-REFERENCES

-   (Smith L. M., et. al AACR 2010 Annual Meeting (abstract #2590);    Gudas J. M., et. al. AACR 2010 Annual Meeting (abstract #4393)

Antibodies

Anti-AGS-5 Antibody: M6.131 (Smith, L. M., et. al AACR 2010 AnnualMeeting (abstract #2590)

(59) ENPP3 (Ectonucleotide pyrophosphatase/phosphodiesterase 3)

Nucleotide

Genbank accession no. AF005632

Genbank version no. AF005632.2 GI:4432589 (SEQ ID NO: 163)

Genbank record update date: Mar. 10, 2010 09:41 PM

Polypeptide

Genbank accession no. AAC51813

Genbank version no. AAC51813.1 GI:2465540 (SEQ ID NO: 164)

Genbank record update date: Mar. 10, 2010 09:41 PM

CROSS-REFERENCES

-   Jin-Hua P., et al Genomics 45 (2), 412-415 (1997)

Other Information

Official Symbol: ENPP3

Other Aliases: RP5-988G15.3, B10, CD203c, NPP3, PD-IBETA, PDNP3

Other Designations: E-NPP 3; dJ1005H11.3 (phosphodiesterase I/nucleotidepyrophosphatase 3); dJ914N13.3 (phosphodiesterase I/nucleotidepyrophosphatase 3); ectonucleotide pyrophosphatase/phosphodiesterasefamily member 3; gp130RB13-6; phosphodiesterase I beta;phosphodiesterase I/nucleotide pyrophosphatase 3; phosphodiesterase-Ibeta

(60) PRR4 (Proline rich 4 (lacrimal))

Nucleotide

Genbank accession no. NM_007244

Genbank version no. NM_007244.2 GI:154448885 (SEQ ID NO: 165)

Genbank record update date: Jun. 28, 2012 12:39 PM

Polypeptide

Genbank accession no. NP_009175

Genbank version no. NP_009175.2 GI:154448886 (SEQ ID NO: 166)

Genbank record update date: Jun. 28, 2012 12:39 PM

CROSS-REFERENCES

-   Dickinson D. P., et al Invest. Ophthalmol. Vis. Sci. 36 (10),    2020-2031 (1995)

Other Information

Official Symbol: PRR4

Other Aliases: LPRP, PROL4

Other Designations: lacrimal proline-rich protein; nasopharyngealcarcinoma-associated proline-rich protein 4; proline-rich polypeptide 4;proline-rich protein 4

(61) GCC—GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor)

Nucleotide

Genbank accession no. NM_004963

Genbank version no. NM_004963.3 GI:222080082 (SEQ ID NO: 167)

Genbank record update date: Sep. 2, 2012 01:50 PM

Polypeptide

Genbank accession no. NP_004954

Genbank version no. NP_004954.2 GI:222080083 (SEQ ID NO: 168)

Genbank record update date: Sep. 2, 2012 01:50 PM

CROSS REFERENCE

-   De Sauvage F. J., et al J. Biol. Chem. 266 (27), 17912-17918 (1991);    Singh S., et al Biochem. Biophys. Res. Commun. 179 (3), 1455-1463    (1991)

Other Information

Official Symbol: GUCY2C

Other Aliases: DIAR6, GUC2C, MUCIL, STAR

Other Designations: GC-C; STA receptor; guanylyl cyclase C; hSTAR;heat-stable enterotoxin receptor; intestinal guanylate cyclase

(62) Liv-1—SLC39A6 (Solute carrier family 39 (zinc transporter), member6)

Nucleotide

Genbank accession no. U41060

Genbank version no. U41060.2 GI:12711792 (SEQ ID NO: 169)

Genbank record update date: Nov. 30, 2009 04:35 PM

Polypeptide

Genbank accession no. AAA96258

Genbank version no. AAA96258.2 GI:12711793 (SEQ ID NO: 170)

Genbank record update date: Nov. 30, 2009 04:35 PM

CROSS-REFERENCES

-   Taylor K M., et al Biochim Biophys Acta. 2003 Apr. 1;    1611(1-2):16-30

Other Information

Official Symbol: SLC39A6

Other Aliases: LIV-1

Other Designations: LIV-1 protein, estrogen regulated; ZIP-6;estrogen-regulated protein LIV-1; solute carrier family 39 (metal iontransporter), member 6; solute carrier family 39 member 6; zinctransporter ZIP6; zrt- and Irt-like protein 6

(63) 5T4, Trophoblast glycoprotein, TPBG—TPBG (trophoblast glycoprotein)

Nucleotide

Genbank accession no. AJ012159

Genbank version no. AJ012159.1 GI:3805946 (SEQ ID NO: 171)

Genbank record update date: Feb. 1, 2011 10:27 AM

Polypeptide

Genbank accession no. CAA09930

Genbank version no. CAA09930.1 GI:3805947 (SEQ ID NO: 172)

Genbank record update date: Feb. 1, 2011 10:27 AM

CROSS-REFERENCES

-   King K. W., et al Biochim. Biophys. Acta 1445 (3), 257-270 (1999)

Other Information

-   -   Official Symbol: TPBG    -   Other Aliases: 5T4, 5T4AG, M6P1    -   Other Designations: 5T4 oncofetal antigen; 5T4 oncofetal        trophoblast glycoprotein; 5T4 oncotrophoblast glycoprotein

(64) CD56—NCMA 1 (Neural cell adhesion molecule 1)

Nucleotide

Genbank accession no. NM_000615

Genbank version no. NM_000615.6 GI:336285433 (SEQ ID NO: 173)

Genbank record update date: Sep. 23, 2012 02:32 PM

Polypeptide

Genbank accession no. NP_000606

Genbank version no. NP_000606.3 GI:94420689 (SEQ ID NO: 174)

Genbank record update date: Sep. 23, 2012 02:32 PM

CROSS-REFERENCES

-   Dickson, G., et al, Cell 50 (7), 1119-1130 (1987)

Other Information

Official Symbol: NCAM1

Other Aliases: CD56, MSK39, NCAM

Other Designations: antigen recognized by monoclonal antibody 5.1H11;neural cell adhesion molecule, NCAM

Antibodies

Immunogen: HuN901 (Smith S V., et al Curr Opin Mol Ther. 2005 August;7(4):394-401)

-   -   For example, see humanized from murine N901 antibody. See FIGS.        1b and 1e of Roguska, M. A., et al. Proc Natl Acad Sci USA        February 1994; 91:969-973.

(65) CanAg (Tumor associated antigen CA242)

CROSS-REFERENCES

-   Haglund C., et al Br J Cancer 60:845-851, 1989; Baeckstrom D., et al    J Biol Chem 266:21537-21547, 1991

Antibodies

huC242 (Tolcher A W et al., J Clin Oncol. 2003 Jan. 15; 21(2):211-22;Immunogen)

-   -   For example, see US20080138898A1 SEQ ID NO: 1 and 2

(66) FOLRI (Folate Receptor 1)

Nucleotide

Genbank accession no. J05013

Genbank version no. J05013.1 GI:182417 (SEQ ID NO: 175)

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA35823

Genbank version no. AAA35823.1 GI:182418 (SEQ ID NO: 176)

Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS-REFERENCES

-   Elwood P. C., et al J. Biol. Chem. 264 (25), 14893-14901 (1989)

Other Information

Official Symbol: FOLR1

Other Aliases: FBP, FOLR

Other Designations: FR-alpha; KB cells FBP; adult folate-bindingprotein; folate binding protein; folate receptor alpha; folate receptor,adult; ovarian tumor-associated antigen MOv18

Antibodies

M9346A—Whiteman K R., et al Cancer Res Apr. 15, 2012; 72(8 Supplement):4628 (Immunogen)

(67) GPNMB (Glycoprotein (transmembrane) nmb)

Nucleotide

Genbank accession no. X76534

Genbank version no. X76534.1 GI:666042 (SEQ ID NO: 177)

Genbank record update date: Feb. 2, 2011 10:10 AM

Polypeptide

Genbank accession no. CAA54044

Genbank version no. CAA54044.1 GI:666043 (SEQ ID NO: 178)

Genbank record update date: Feb. 2, 2011 10:10 AM

CROSS-REFERENCES

-   Weterman M. A., et al Int. J. Cancer 60 (1), 73-81 (1995)

Other Information

Official Symbol: GPNMB

Other Aliases: UNQ1725/PRO9925, HGFIN, NMB

Other Designations: glycoprotein NMB; glycoprotein nmb-like protein;osteoactivin; transmembrane glycoprotein HGFIN; transmembraneglycoprotein NMB

Antibodies

-   Celldex Therapeutics: CR011 (Tse K F., et al Clin Cancer Res. 2006    Feb. 15; 12(4):1373-82)    -   For example, see EP1827492B1 SEQ ID NO: 22, 24, 26, 31, 33 and        35

(68) TIM-1—HA VCR (Hepatitis A virus cellular receptor 1)

Nucleotide

Genbank accession no. AF043724

Genbank version no. AF043724.1 GI:2827453 (SEQ ID NO: 179)

Genbank record update date: Mar. 10, 2010 06:24 PM

Polypeptide

Genbank accession no. AAC39862

Genbank version no. AAC39862.1 GI:2827454 (SEQ ID NO: 180)

Genbank record update date: Mar. 10, 2010 06:24 PM

CROSS-REFERENCES

-   Feigelstock D., et al J. Virol. 72 (8), 6621-6628 (1998)

Other Information

Official Symbol: HAVCR1

Other Aliases: HAVCR, HAVCR-1, KIM-1, KIM1, TIM, TIM-1, TIM1, TIMD-1,TIMD1

Other Designations: T cell immunoglobin domain and mucin domain protein1; T-cell membrane protein 1; kidney injury molecule 1

(69) RG-1/Prostate tumor target Mindin—Mindin/RG-1

CROSS-REFERENCES

-   Parry R., et al Cancer Res. 2005 Sep. 15; 65(18):8397-405

(70) B7-H4—VTCN1 (V-set domain containing T cell activation inhibitor 1

Nucleotide

Genbank accession no. BX648021

Genbank version no. BX648021.1 GI:34367180 (SEQ ID NO: 181)

Genbank record update date: Feb. 2, 2011 08:40 AM

CROSS-REFERENCES

-   Sica G L., et al Immunity. 2003 June; 18(6):849-61

Other Information

Official Symbol: VTCN1

Other Aliases: RP11-229A19.4, B7-H4, B7H4, B7S1, B7X, B7h.5, PRO1291,VCTN1

Other Designations: B7 family member, H4; B7 superfamily member 1; Tcell costimulatory molecule B7x; T-cell costimulatory molecule B7x;V-set domain-containing T-cell activation inhibitor 1; immunecostimulatory protein B7-H4

(71) PTK7 (PTK7 protein tyrosine kinase 7)

Nucleotide

Genbank accession no. AF447176

Genbank version no. AF447176.1 GI:17432420 (SEQ ID NO: 182)

Genbank record update date: Nov. 28, 2008 01:51 PM

Polypeptide

Genbank accession no. AAL39062

Genbank version no. AAL39062.1 GI:17432421 (SEQ ID NO: 183)

Genbank record update date: Nov. 28, 2008 01:51 PM

CROSS-REFERENCES

-   Park S. K., et al J. Biochem. 119 (2), 235-239 (1996)

Other Information

Official Symbol: PTK7

Other Aliases: CCK-4, CCK4

Other Designations: colon carcinoma kinase 4; inactive tyrosine-proteinkinase 7; pseudo tyrosine kinase receptor 7; tyrosine-proteinkinase-like 7

(72) CD37 (CD37 molecule)

Nucleotide

Genbank accession no. NM_001040031

Genbank version no. NM_001040031.1 GI:91807109 (SEQ ID NO: 184)

Genbank record update date: Jul. 29, 2012 02:08 PM

Polypeptide

Genbank accession no. NP_001035120

Genbank version no. NP_001035120.1 GI:91807110 (SEQ ID NO: 185)

Genbank record update date: Jul. 29, 2012 02:08 PM

CROSS-REFERENCES

-   Schwartz-Albiez R., et al J. Immunol. 140 (3), 905-914 (1988)

Other information

Official Symbol: CD37

Other Aliases: GP52-40, TSPAN26

Other Designations: CD37 antigen; cell differentiation antigen 37;leukocyte antigen CD37; leukocyte surface antigen CD37; tetraspanin-26;tspan-26

Antibodies

-   Boehringer Ingelheim: mAb 37.1 (Heider K H., et al Blood. 2011 Oct.    13; 118(15):4159-68)-   Trubion: CD37-SMIP (G28-1 scFv-lg) ((Zhao X., et al Blood. 2007;    110: 2569-2577)    -   For example, see US20110171208A1 SEQ ID NO: 253-   Immunogen: K7153A (Deckert J., et al Cancer Res Apr. 15, 2012; 72(8    Supplement): 4625)

(73) CD138-SDC1 (syndecan 1)

Nucleotide

Genbank accession no. AJ551176

Genbank version no. AJ551176.1 GI:29243141 (SEQ ID NO: 186)

Genbank record update date: Feb. 1, 2011 12:09 PM

Polypeptide

Genbank accession no. CAD80245

Genbank version no. CAD80245.1 GI:29243142 (SEQ ID NO: 187)

Genbank record update date: Feb. 1, 2011 12:09 PM

CROSS-REFERENCES

-   O'Connell F P., et al Am J Clin Pathol. 2004 February; 121(2):254-63

Other Information

Official Symbol: SDC1

Other Aliases: CD138, SDC, SYND1, syndecan

Other Designations: CD138 antigen; heparan sulfate proteoglycanfibroblast growth factor receptor; syndecan proteoglycan 1; syndecan-1

Antibodies

-   Biotest: chimerized MAb (nBT062)—(Jagannath S., et al Poster ASH    #3060, 2010; WIPO Patent Application WO/2010/128087)    -   For example, see US20090232810 SEQ ID NO: 1 and 2-   Immunogen: B-B4 (Tassone P., et al Blood 104_3688-3696)    -   For example, see US20090175863A1 SEQ ID NO: 1 and 2

(74) CD74 (CD74 molecule, major histocompatibility complex, class IIinvariant chain)

Nucleotide

Genbank accession no. NM_004355

Genbank version no. NM_004355.1 GI:343403784 (SEQ ID NO: 188)

Genbank record update date: Sep. 23, 2012 02:30 PM

Polypeptide

Genbank accession no. NP_004346

Genbank version no. NP_004346.1 GI:10835071 (SEQ ID NO: 189)

Genbank record update date: Sep. 23, 2012 02:30 PM

CROSS-REFERENCES

-   Kudo, J., et al Nucleic Acids Res. 13 (24), 8827-8841 (1985)

Other Information

Official Symbol: CD74

Other Aliases: DHLAG, HLADG, II, la-GAMMA

Other Designations: CD74 antigen (invariant polypeptide of majorhistocompatibility complex, class II antigen-associated); HLA class IIhistocompatibility antigen gamma chain; HLA-DR antigens-associatedinvariant chain; HLA-DR-gamma; la-associated invariant chain; MHC HLA-DRgamma chain; gamma chain of class II antigens; p33

Antibodies

-   Immunomedics: hLL1 (Milatuzumab)—Berkova Z., et al Expert Opin    Investig Drugs. 2010 January; 19(1):141-9)    -   For example, see US20040115193 SEQ ID NOs: 19, 20, 21, 22, 23        and 24

Genmab: HuMax-CD74 (see website)

(75) Claudins—CLs (Claudins)

CROSS-REFERENCES

-   Offner S., et al Cancer Immunol Immunother. 2005 May; 54(5):431-45,    Suzuki H., et al Ann N Y Acad Sci. 2012 July; 1258:65-70)

In humans, 24 members of the family have been described—see literaturereference.

(76) EGFR (Epidermal growth factor receptor)

Nucleotide

Genbank accession no. NM_005228

Genbank version no. NM_005228.3 GI:41927737 (SEQ ID NO:190)(GI:41327737)

Genbank record update date: Sep. 30, 2012 01:47 PM

Polypeptide

Genbank accession no. NP_005219

Genbank version no. NP_005219.2 GI:29725609 (SEQ ID NO: 191)

Genbank record update date: Sep. 30, 2012 01:47 PM

CROSS-REFERENCES

-   Dhomen N S., et al Crit Rev Oncog. 2012; 17(1):31-50

Other Information

Official Symbol: EGFR

Other Aliases: ERBB, ERBB1, HER1, PIG61, mENA

Other Designations: avian erythroblastic leukemia viral (v-erb-b)oncogene homolog; cell growth inhibiting protein 40; cellproliferation-inducing protein 61; proto-oncogene c-ErbB-1; receptortyrosine-protein kinase erbB-1

Antibodies

-   BMS: Cetuximab (Erbitux)—Broadbridge V T., et al Expert Rev    Anticancer Ther. 2012 May; 12(5):555-65.    -   For example, see U.S. Pat. No. 6,217,866—ATTC deposit No. 9764.-   Amgen: Panitumumab (Vectibix)—Argiles G., et al Future Oncol. 2012    April; 8(4):373-89    -   For example, see U.S. Pat. No. 6,235,883 SEQ ID NOs: 23-38.-   Genmab: Zalutumumab—Rivera F., et al Expert Opin Biol Ther. 2009    May; 9(5):667-74.-   YM Biosciences: Nimotuzumab—Ramakrishnan M S., et al MAbs. 2009    January-February; 1(1):41-8.    -   For example, see U.S. Pat. No. 5,891,996 SEQ ID NOs: 27-34.

(77) Her3 (ErbB3)—ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogenehomolog 3 (avian))

Nucleotide

Genbank accession no. M34309

Genbank version no. M34309.1 GI:183990 (SEQ ID NO: 192)

Genbank record update date: Jun. 23, 2010 08:47 PM

Polypeptide

Genbank accession no. AAA35979

Genbank version no. AAA35979.1 GI:306841 (SEQ ID NO: 193)

Genbank record update date: Jun. 23, 2010 08:47 PM

CROSS-REFERENCES

-   Plowman, G. D., et al., Proc. Natl. Acad. Sci. U.S.A. 87 (13),    4905-4909 (1990)

Other Information

Official Symbol: ERBB3

Other Aliases: ErbB-3, HER3, LCCS2, MDA-BF-1, c-erbB-3, c-erbB3,erbB3-S, p180-ErbB3, p45-sErbB3, p85-sErbB3

Other Designations: proto-oncogene-like protein c-ErbB-3; receptortyrosine-protein kinase erbB-3; tyrosine kinase-type cell surfacereceptor HER3

Antibodies

-   Merimack Pharma: MM-121 (Schoeberl B., et al Cancer Res. 2010 Mar.    15; 70(6):2485-2494)    -   For example, see US2011028129 SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7        and 8.

(78) RON—MSTIR (macrophage stimulating 1 receptor (c-met-relatedtyrosine kinase))

Nucleotide

Genbank accession no. X70040

Genbank version no. X70040.1 GI:36109 (SEQ ID NO: 194)

Genbank record update date: Feb. 2, 2011 10:17 PM

Polypeptide

Genbank accession no. CCA49634

Genbank version no. CCA49634.1 GI:36110 (SEQ ID NO: 195)

Genbank record update date: Feb. 2, 2011 10:17 PM

CROSS-REFERENCES

-   Ronsin C., et al Oncogene 8 (5), 1195-1202 (1993)

Other Information

Official Symbol: MST1R

Other Aliases: CD136, CDw136, PTK8, RON

Other Designations: MSP receptor; MST1R variant RON30; MST1R variantRON62; PTK8 protein tyrosine kinase 8; RON variant E2E3; c-met-relatedtyrosine kinase; macrophage-stimulating protein receptor; p185-Ron;soluble RON variant 1; soluble RON variant 2; soluble RON variant 3;soluble RONvariant 4

(79) EPHA2 (EPH receptor A2)

Nucleotide

Genbank accession no. BC037166

Genbank version no. BC037166.2 GI:33879863 (SEQ ID NO: 196)

Genbank record update date: Mar. 6, 2012 01:59 PM

Polypeptide

Genbank accession no. AAH37166

Genbank version no. AAH37166.1 GI:22713539 (SEQ ID NO: 197)

Genbank record update date: Mar. 6, 2012 01:59 PM

CROSS-REFERENCES

-   Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99 (26),    16899-16903 (2002)

Other Information

Official Symbol: EPHA2

Other Aliases: ARCC2, CTPA, CTPP1, ECK

Other Designations: ephrin type-A receptor 2; epithelial cell receptorprotein tyrosine kinase; soluble EPHA2 variant 1; tyrosine-proteinkinase receptor ECK

Antibodies

-   Medimmune: 1C1 (Lee J W., et al Clin Cancer Res. 2010 May 1;    16(9):2562-2570)    -   For example, see US20090304721A1 FIGS. 7 and 8.

(80) CD20—MS4A1 (membrane-spanning 4-domains, subfamily A, member 1)

Nucleotide

Genbank accession no. M27394

Genbank version no. M27394.1 GI:179307 (SEQ ID NO: 198)

Genbank record update date: Nov. 30, 2009 11:16 AM

Polypeptide

Genbank accession no. AAA35581

Genbank version no. AAA35581.1 GI:179308 (SEQ ID NO: 199)

Genbank record update date: Nov. 30, 2009 11:16 AM

CROSS-REFERENCES

-   Tedder T. F., et al Proc. Natl. Acad. Sci. U.S.A. 85 (1), 208-212    (1988)

Other Information

Official Symbol: MS4A1

Other Aliases: B1, Bp35, CD20, CVID5, LEU-16, MS4A2, S7

Other Designations: B-lymphocyte antigen CD20; B-lymphocyte cell-surfaceantigen B1; CD20 antigen; CD20 receptor; leukocyte surface antigenLeu-16

Antibodies

-   Genentech/Roche: Rituximab—Abdulla N E., et al BioDrugs. 2012 Apr.    1; 26(2):71-82.    -   For example, see U.S. Pat. No. 5,736,137, ATCC deposit No.        HB-69119.

GSK/Genmab: Ofatumumab—Nightingale G., et al Ann Pharmacother. 2011October; 45(10):1248-55.

-   -   For example, see US20090169550A1 SEQ ID NOs: 2, 4 and 5.

-   Immunomedics: Veltuzumab—Goldenberg D M., et al Leuk Lymphoma. 2010    May; 51(5):747-55.    -   For example, see U.S. Pat. No. 7,919,273B2 SEQ ID NOs: 1, 2, 3,        4, 5 and 6.

(81) Tenascin C—TNC (Tenascin C)

Nucleotide

Genbank accession no. NM_002160

Genbank version no. NM_002160.3 GI:340745336 (SEQ ID NO: 200)

Genbank record update date: Sep. 23, 2012 02:33 PM

Polypeptide

Genbank accession no. NP_002151

Genbank version no. NP_002151.2 GI:153946395 (SEQ ID NO: 201)

Genbank record update date: Sep. 23, 2012 02:33 PM

CROSS-REFERENCES

-   Nies D. E., et al J. Biol. Chem. 266 (5), 2818-2823 (1991); Siri A.,    et al Nucleic Acids Res. 19 (3), 525-531 (1991)

Other information

Official Symbol: TNC

Other Aliases: 150-225, GMEM, GP, HXB, JI, TN, TN-C

Other Designations: GP 150-225; cytotactin;glioma-associated-extracellular matrix antigen; hexabrachion (tenascin);myotendinous antigen; neuronectin; tenascin; tenascin-C isoform 14/AD1/16

Antibodies

-   Philogen: G11 (von Lukowicz T., et al J Nucl Med. 2007 April;    48(4):582-7) and F16 (Pedretti M., et al Lung Cancer. 2009 April;    64(1):28-33)    -   For example, see U.S. Pat. No. 7,968,685 SEQ ID NOs: 29, 35, 45        and 47.

(82) FAP (Fibroblast activation protein, alpha)

Nucleotide

Genbank accession no. U09278

Genbank version no. U09278.1 GI:1888315 (SEQ ID NO: 202)

Genbank record update date: Jun. 23, 2010 09:22 AM

Polypeptide

Genbank accession no. AAB49652

Genbank version no. AAB49652.1 GI:1888316 (SEQ ID NO: 203)

Genbank record update date: Jun. 23, 2010 09:22 AM

CROSS REFERENCE

-   Scanlan, M. J., et al Proc. Natl. Acad. Sci. U.S.A. 91 (12),    5657-5661 (1994)

Other information

Official Symbol: FAP

Other Aliases: DPPIV, FAPA

Other Designations: 170 kDa melanoma membrane-bound gelatinase; integralmembrane serine protease; seprase

(83) DKK-1 (Dickkopf 1 homolog (Xenopus laevis)

Nucleotide

Genbank accession no. NM_012242

Genbank version no. NM_012242.2 GI:61676924 (SEQ ID NO: 204)

Genbank record update date: Sep. 30, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_036374

Genbank version no. NP_036374.1 GI:7110719 (SEQ ID NO: 205)

Genbank record update date: Sep. 30, 2012 01:48 PM

CROSS-REFERENCES

-   Fedi P. et al J. Biol. Chem. 274 (27), 19465-19472 (1999)

Other information

Official Symbol: DKK1

Other Aliases: UNQ492/PRO1008, DKK-1, SK

Other Designations: dickkopf related protein-1; dickkopf-1 like;dickkopf-like protein 1; dickkopf-related protein 1; hDkk-1

Antibodies

-   Novartis: BHQ880 (Fulciniti M., et al Blood. 2009 Jul. 9;    114(2):371-379)

For example, see US20120052070A1 SEQ ID NOs: 100 and 108.

(84) CD52 (CD52 molecule)

Nucleotide

Genbank accession no. NM_001803

Genbank version no. NM_001803.2 GI:68342029 (SEQ ID NO: 206)

Genbank record update date: Sep. 30, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_001794

Genbank version no. NP_001794.2 GI:68342030 (SEQ ID NO: 207)

Genbank record update date: Sep. 30, 2012 01:48 PM

CROSS-REFERENCES

-   Xia M. Q., et al Eur. J. Immunol. 21 (7), 1677-1684 (1991)

Other Information

Official Symbol: CD52

Other Aliases: CDW52

Other Designations: CAMPATH-1 antigen; CD52 antigen (CAMPATH-1 antigen);CDW52 antigen (CAMPATH-1 antigen); cambridge pathology 1 antigen;epididymal secretory protein E5; he5; human epididymis-specific protein5

Antibodies

-   Alemtuzumab (Campath)—Skoetz N., et al Cochrane Database Syst Rev.    2012 Feb. 15; 2:CD008078.

For example, see Drugbank Acc. No. DB00087 (BIOD00109, BTD00109)

(85) CS1—SLAMF7 (SLAM family member 7)

Nucleotide

Genbank accession no. NM_021181

Genbank version no. NM_021181.3 GI:1993571 (SEQ ID NO:208)(**GI:19923571)

Genbank record update date: Jun. 29, 2012 11:24 AM

Polypeptide

Genbank accession no. NP_067004

Genbank version no. NP_067004.3 GI:19923572 (SEQ ID NO: 209)

Genbank record update date: Jun. 29, 2012 11:24 AM

CROSS-REFERENCES

-   Boles K. S., et al Immunogenetics 52 (3-4), 302-307 (2001)

Other Information

Official Symbol: SLAMF7

Other Aliases: UNQ576/PRO1138, 19A, CD319, CRACC, CS1

Other Designations: 19A24 protein; CD2 subset 1; CD2-like receptoractivating cytotoxic cells; CD2-like receptor-activating cytotoxiccells; membrane protein FOAP-12; novel LY9 (lymphocyte antigen 9) likeprotein; protein 19A

Antibodies

BMS: elotuzumab/HuLuc63 (Benson D M., et al J Clin Oncol. 2012 Jun. 1;30(16):2013-2015) For example, see US20110206701 SEQ ID NOs: 9, 10, 11,12, 13, 14, 15 and 16.

(86) Endoglin—ENG (Endoglin)

Nucleotide

Genbank accession no. AF035753

Genbank version no. AF035753.1 GI:3452260 (SEQ ID NO: 210)

Genbank record update date: Mar. 10, 2010 06:36 PM

Polypeptide

Genbank accession no. AAC32802

Genbank version no. AAC32802.1 GI:3452261 (SEQ ID NO: 211)

Genbank record update date: Mar. 10, 2010 06:36 PM

CROSS-REFERENCES

-   Rius C., et al Blood 92 (12), 4677-4690 (1998)

Official Symbol: ENG

Other Information

Other Aliases: RP11-228B15.2, CD105, END, HHT1, ORW, ORW1

Other Designations: CD105 antigen

(87) Annexin A1—ANXA1 (Annexin A1)

Nucleotide

Genbank accession no. X05908

Genbank version no. X05908.1 GI:34387 (SEQ ID NO: 212)

Genbank record update date: Feb. 2, 2011 10:02 AM

Polypeptide

Genbank accession no. CCA29338

Genbank version no. CCA29338.1 GI:34388 (SEQ ID NO: 213)

Genbank record update date: Feb. 2, 2011 10:02 AM

CROSS-REFERENCES

-   Wallner B. P., et al Nature 320 (6057), 77-81 (1986)

Other Information

Official Symbol: ANXA1

Other Aliases: RP11-71A24.1, ANX1, LPC1

Other Designations: annexin I (lipocortin I); annexin-1; calpactin II;calpactin-2; chromobindin-9; lipocortin I; p35; phospholipase A2inhibitory protein

(88) V-CAM (CD106)—VCAM1 (Vascular cell adhesion molecule 1)

Nucleotide

Genbank accession no. M60335

Genbank version no. M60335.1 GI:340193 (SEQ ID NO: 214)

Genbank record update date: Jun. 23, 2010 08:56 AM

Polypeptide

Genbank accession no. AAA61269

Genbank version no. AAA61269.1 GI:340194 (SEQ ID NO: 215)

Genbank record update date: Jun. 23, 2010 08:56 AM

CROSS-REFERENCES

-   Hession C., et al J. Biol. Chem. 266 (11), 6682-6685 (1991)

Other Information

Official Symbol VCAM1

Other Aliases: CD106, INCAM-100

Other Designations: CD106 antigen; vascular cell adhesion protein 1

RHAB6.2 (SEQ ID NO: 3)QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTEYAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTAVPNLRGDLQVLAQKVAGPYPFDYWGQGTLVTVSS RHCB6.2 (SEQ ID NO: 4)QVQLVQSGAEVKKPGASVKVSCKASGYTFIDSYMHWVRQAPGQRLEWMGWIDPENGDTEYAPKFQGRVTITTDTSASTAYMELSSLRSEDTAVYYCARGTPTAVPNLRGDLQVLAQKVAGPYPFDYWGQGTLVTVSS RHF (SEQ ID NO: 5)QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGDTEYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTGPYYFDYWGQGTLVTV SS RHFB6(SEQ ID NO: 6)QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGDTEYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTAVPNLRGDLQVLAQKVAGPYYFDYWGQGTLVTVSS RHAY100bP (SEQ ID NO: 7)QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTEYAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTGPYPFDYWGQGTLVTVSS RKF(SEQ ID NO: 8)ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RKFL36L50 (SEQ ID NO: 9)ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWLQQKPGQAPRLLIYLTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RKC (SEQ ID NO: 10)EIVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK Anti-CD33 CD33 Hum195 VH(SEQ ID NO: 11)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS CD33 Hum195 VK(SEQ ID NO: 12)DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK Anti-CD19CD19 B4 resurfaced VH (SEQ ID NO: 13)QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQRPGQGLEWIGEIDPSDSYTNYNQNFKGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMDYWGQGTSVTV SSCD19 B4 resurfaced VK (SEQ ID NO: 14)EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTKLEIK Anti-Her2Herceptin VH chain (SEQ ID NO: 15)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SHerceptin VL chain (SEQ ID NO: 16)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK Anti-CD25Simulect VK (also known as Basiliximab) (SEQ ID NO: 17)QIVSTQSPAIMSASPGEKVTMTCSASSSRSYMQWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYTFGGGTKLEIK Simulect VH (SEQ ID NO: 18)QLQQSGTVLARPGASVKMSCKASGYSFTRYWMHWIKQRPGQGLEWIGAIYPGNSDTSYNQKFEGKAKLTAVTSASTAYMELSSLTHEDSAVYYCSRDYGYYFDFWGQGTTLTVSS Anti-PSMADeimmunised VH ‘1 (SEQ ID NO: 19)EVQLVQSGPEVKKPGATVKISCKTSGYTFTEYTIHWVKQAPGKGLEWIGNINPNNGGTTYNQKFEDKATLTVDKSTDTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLLTVSS Deimmunised VK ‘1(SEQ ID NO: 20)DIQMTQSPSSLSTSVGDRVTLTCKASQDVGTAVDWYQQKPGPSPKLLIYYWASTRHTGIPSRFSGSGSGTDFTLTISSLQPEDFADYYCQQYNSYPLTFGPGTKVDIK Deimmunised VH1 ‘5(SEQ ID NO: 21)EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTGVYYCTRRWNNFWGQGTTVTVSSDeimmunised VH2 ‘5 (SEQ ID NO: 22)EVKLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSSDeimmunised VH3 ‘5 (SEQ ID NO: 23)EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSSDeimmunised VH4 ‘5 (SEQ ID NO: 24)EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRFTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSSDeimmunised VK1 ‘5 (SEQ ID NO: 25)NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFTGSGSATDFTLTISSLQTEDLADYYCGQSYTFPYTFGQGTKLEMK Deimmunised VK2 ‘5(SEQ ID NO: 26)NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK3 ‘5(SEQ ID NO: 27)NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK4 ‘5(SEQ ID NO: 28)NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFSGSGSGTDFTLTISSLQAEDEADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK DI ‘5(SEQ ID NO: 29)NIVMTQFPKSMSASAGERMTLTCKASENVGTYVSWYQQKPTQSPKMLIYGASNRFTGVPDRFSGSGSGTDFILTISSVQAEDLVDYYCGQSYTFPYTFGGGTKLEMK Deimmunised VH DI ‘5(SEQ ID NO: 30)EVKLEESGGGLVQPGGSMKISCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKSSVYLQMNSLRAEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHA ‘5 (SEQ ID NO: 31)EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNFATHYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHB ‘5 (SEQ ID NO: 32)EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHC ‘5 (SEQ ID NO: 33)EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHD ‘5 (SEQ ID NO: 34)EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVNRQASGKGLEWVGEIRSQSNNFATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHE ‘5 (SEQ ID NO: 35)EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATHYAESVKGRFTISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHF ‘5 (SEQ ID NO: 36)EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHG ‘5 (SEQ ID NO: 37)EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RKA ‘5 (SEQ ID NO: 38)DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKB ‘5(SEQ ID NO: 39)DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSVVYQQKPGTAPKWYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKC ‘5(SEQ ID NO: 40)DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKD ‘5(SEQ ID NO: 41)DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKE ‘5(SEQ ID NO: 42)NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPDRFTGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKF ‘5(SEQ ID NO: 43)NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFSGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKG ‘5(SEQ ID NO: 44)NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPDRFTGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

The parent antibody may also be a fusion protein comprising analbumin-binding peptide (ABP) sequence (Dennis et al. (2002) “AlbuminBinding As A General Strategy For Improving The Pharmacokinetics OfProteins” J Biol Chem. 277:35035-35043; WO 01/45746). Antibodies of theinvention include fusion proteins with ABP sequences taught by: (i)Dennis et al (2002) J Biol Chem. 277:35035-35043 at Tables III and IV,page 35038; (ii) US 2004/0001827 at [0076]; and (iii) WO 01/45746 atpages 12-13, and all of which are incorporated herein by reference.

In one embodiment, the antibody has been raised to target specific thetumour related antigen α_(v)β₆.

The cell binding agent may be labelled, for example to aid detection orpurification of the agent either prior to incorporation as a conjugate,or as part of the conjugate. The label may be a biotin label. In anotherembodiment, the cell binding agent may be labelled with a radioisotope.

Embodiments of the present invention include ConjA wherein the cellbinding agent is selected from an antibody to any of the antigensdiscussed above.

Embodiments of the present invention include ConjB wherein the cellbinding agent is selected from an antibody to any of the antigensdiscussed above.

Embodiments of the present invention include ConjA wherein the cellbinding agent is selected from any of the antibodies discussed above.

Embodiments of the present invention include ConjB wherein the cellbinding agent is selected from any of the antibodies discussed above.

The present invention may also relate to conjugates where the cellbinding agent is selected from an antibody to any of the antigensdiscussed above and any of the antibodies discussed above linked todifferent drugs.

Drug Loading

The drug loading is the average number of PBD drugs per cell bindingagent, e.g. antibody. Where the compounds of the invention are bound tocysteines, drug loading may range from 1 to 8 drugs (D) per cell bindingagent, i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties arecovalently attached to the cell binding agent. Compositions ofconjugates include collections of cell binding agents, e.g. antibodies,conjugated with a range of drugs, from 1 to 8.

Where the compounds of the invention are bound to lysines, drug loadingmay range from 1 to 80 drugs (D) per cell binding agent, although anupper limit of 40, 20, 10 or 8 may be preferred. Compositions ofconjugates include collections of cell binding agents, e.g. antibodies,conjugated with a range of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to10 or 1 to 8.

The average number of drugs per antibody in preparations of ADC fromconjugation reactions may be characterized by conventional means such asUV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, andelectrophoresis. The quantitative distribution of ADC in terms of p mayalso be determined. By ELISA, the averaged value of p in a particularpreparation of ADC may be determined (Hamblett et al (2004) Clin. CancerRes. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852).However, the distribution of p (drug) values is not discernible by theantibody-antigen binding and detection limitation of ELISA. Also, ELISAassay for detection of antibody-drug conjugates does not determine wherethe drug moieties are attached to the antibody, such as the heavy chainor light chain fragments, or the particular amino acid residues. In someinstances, separation, purification, and characterization of homogeneousADC where p is a certain value from ADC with other drug loadings may beachieved by means such as reverse phase HPLC or electrophoresis. Suchtechniques are also applicable to other types of conjugates.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, an antibody may have onlyone or several cysteine thiol groups, or may have only one or severalsufficiently reactive thiol groups through which a linker may beattached. Higher drug loading, e.g. p>5, may cause aggregation,insolubility, toxicity, or loss of cellular permeability of certainantibody-drug conjugates.

Typically, fewer than the theoretical maximum of drug moieties areconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with thedrug-linker intermediate (D-L) or linker reagent. Only the most reactivelysine groups may react with an amine-reactive linker reagent. Also,only the most reactive cysteine thiol groups may react with athiol-reactive linker reagent. Generally, antibodies do not containmany, if any, free and reactive cysteine thiol groups which may belinked to a drug moiety. Most cysteine thiol residues in the antibodiesof the compounds exist as disulfide bridges and must be reduced with areducing agent such as dithiothreitol (DTT) or TCEP, under partial ortotal reducing conditions. The loading (drug/antibody ratio) of an ADCmay be controlled in several different manners, including: (i) limitingthe molar excess of drug-linker intermediate (D-L) or linker reagentrelative to antibody, (ii) limiting the conjugation reaction time ortemperature, and (iii) partial or limiting reductive conditions forcysteine thiol modification.

Certain antibodies have reducible interchain disulfides, i.e. cysteinebridges. Antibodies may be made reactive for conjugation with linkerreagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by engineering one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues). U.S. Pat. No. 7,521,541teaches engineering antibodies by introduction of reactive cysteineamino acids.

Cysteine amino acids may be engineered at reactive sites in an antibodyand which do not form intrachain or intermolecular disulfide linkages(Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al(2009) Blood 114(13):2721-2729; U.S. Pat. Nos. 7,521,541; 7,723,485;WO2009/052249). The engineered cysteine thiols may react with linkerreagents or the drug-linker reagents of the present invention which havethiol-reactive, electrophilic groups such as maleimide or alpha-haloamides to form ADC with cysteine engineered antibodies and the PBD drugmoieties. The location of the drug moiety can thus be designed,controlled, and known. The drug loading can be controlled since theengineered cysteine thiol groups typically react with thiol-reactivelinker reagents or drug-linker reagents in high yield. Engineering anIgG antibody to introduce a cysteine amino acid by substitution at asingle site on the heavy or light chain gives two new cysteines on thesymmetrical antibody. A drug loading near 2 can be achieved with nearhomogeneity of the conjugation product ADC.

Where more than one nucleophilic or electrophilic group of the antibodyreacts with a drug-linker intermediate, or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of drug moieties attached to an antibody,e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymericreverse phase (PLRP) and hydrophobic interaction (HIC) may separatecompounds in the mixture by drug loading value. Preparations of ADC witha single drug loading value (p) may be isolated, however, these singleloading value ADCs may still be heterogeneous mixtures because the drugmoieties may be attached, via the linker, at different sites on theantibody.

Thus the antibody-drug conjugate compositions of the invention includemixtures of antibody-drug conjugate compounds where the antibody has oneor more PBD drug moieties and where the drug moieties may be attached tothe antibody at various amino acid residues.

In one embodiment, the average number of dimer pyrrolobenzodiazepinegroups per cell binding agent is in the range 1 to 20. In someembodiments the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4,and 4 to 8.

In some embodiments, there is one dimer pyrrolobenzodiazepine group percell binding agent.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as wellas conventional protected forms. Similarly, a reference to an aminogroup includes the protonated form (—N⁺HR¹R²), a salt or solvate of theamino group, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO), then a salt may be formed witha suitable cation. Examples of suitable inorganic cations include, butare not limited to, alkali metal ions such as Na₄ ⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺,NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammoniumions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic acidand valeric. Examples of suitable polymeric organic anions include, butare not limited to, those derived from the following polymeric acids:tannic acid, carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

The invention includes compounds where a solvent adds across the iminebond of the PBD moiety, which is illustrated below where the solvent iswater or an alcohol (R^(A)OH, where R^(A) is C₁₋₄ alkyl):

These forms can be called the carbinolamine and carbinolamine etherforms of the PBD (as described in the section relating to R¹⁰ above).The balance of these equilibria depend on the conditions in which thecompounds are found, as well as the nature of the moiety itself.

These particular compounds may be isolated in solid form, for example,by lyophilisation.

Isomers

Certain compounds of the invention may exist in one or more particulargeometric, optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and I-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and ß-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand I or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or I meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Examples of isotopes that can be incorporated into compounds of theinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, and chlorine, such as, but not limited to ²H(deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S,³⁶Cl, and ¹²⁵I. Various isotopically labeled compounds of the presentinvention, for example those into which radioactive isotopes such as 3H,13C, and 14C are incorporated. Such isotopically labelled compounds maybe useful in metabolic studies, reaction kinetic studies, detection orimaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. Deuterium labelled or substituted therapeutic compounds of theinvention may have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism, and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements. An18F labeled compound may be useful for PET or SPECT studies.Isotopically labeled compounds of this invention and prodrugs thereofcan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. Further, substitution with heavierisotopes, particularly deuterium (i.e., 2H or D) may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements or animprovement in therapeutic index. It is understood that deuterium inthis context is regarded as a substituent. The concentration of such aheavier isotope, specifically deuterium, may be defined by an isotopicenrichment factor. In the compounds of this invention any atom notspecifically designated as a particular isotope is meant to representany stable isotope of that atom.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Biological Activity

In Vitro Cell Proliferation Assays

Generally, the cytotoxic or cytostatic activity of an antibody-drugconjugate (ADC) is measured by: exposing mammalian cells having receptorproteins, e.g. HER2, to the antibody of the ADC in a cell culturemedium; culturing the cells for a period from about 6 hours to about 5days; and measuring cell viability. Cell-based in vitro assays are usedto measure viability (proliferation), cytotoxicity, and induction ofapoptosis (caspase activation) of an ADC of the invention.

The in vitro potency of antibody-drug conjugates can be measured by acell proliferation assay. The CellTiter-Glo® Luminescent Cell ViabilityAssay is a commercially available (Promega Corp., Madison, Wis.),homogeneous assay method based on the recombinant expression ofColeoptera luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713 and5,700,670). This cell proliferation assay determines the number ofviable cells in culture based on quantitation of the ATP present, anindicator of metabolically active cells (Crouch et al (1993) J. Immunol.Meth. 160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay isconducted in 96 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing. The cells may be treated continuously with ADC, or they may betreated and separated from ADC. Generally, cells treated briefly, i.e. 3hours, showed the same potency effects as continuously treated cells.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons.

The in vitro potency of antibody-drug conjugates can also be measured bya cytotoxicity assay. Cultured adherent cells are washed with PBS,detached with trypsin, diluted in complete medium, containing 10% FCS,centrifuged, re-suspended in fresh medium and counted with ahaemocytometer. Suspension cultures are counted directly. Monodispersecell suspensions suitable for counting may require agitation of thesuspension by repeated aspiration to break up cell clumps.

The cell suspension is diluted to the desired seeding density anddispensed (1001 per well) into black 96 well plates. Plates of adherentcell lines are incubated overnight to allow adherence. Suspension cellcultures can be used on the day of seeding.

A stock solution (1 ml) of ADC (20 μg/ml) is made in the appropriatecell culture medium. Serial 10-fold dilutions of stock ADC are made in15 ml centrifuge tubes by serially transferring 100 μl to 900 μl of cellculture medium.

Four replicate wells of each ADC dilution (100 μl) are dispensed in96-well black plates, previously plated with cell suspension (100 μl),resulting in a final volume of 200 μl1. Control wells receive cellculture medium (100 μl).

If the doubling time of the cell line is greater than 30 hours, ADCincubation is for 5 days, otherwise a four day incubation is done.

At the end of the incubation period, cell viability is assessed with theAlamar blue assay. AlamarBlue (Invitrogen) is dispensed over the wholeplate (20 μl per well) and incubated for 4 hours. Alamar bluefluorescence is measured at excitation 570 nm, emission 585 nm on theVarioskan flash plate reader. Percentage cell survival is calculatedfrom the mean fluorescence in the ADC treated wells compared to the meanfluorescence in the control wells.

In Vivo Efficacy

The in vivo efficacy of antibody-drug conjugates (ADC) of the inventioncan be measured by tumor xenograft studies in mice. For example, the invivo efficacy of an anti-HER2 ADC of the invention can be measured by ahigh expressing HER2 transgenic explant mouse model. An allograft ispropagated from the Fo5 mmtv transgenic mouse which does not respond to,or responds poorly to, HERCEPTIN® therapy. Subjects are treated oncewith ADC at certain dose levels (mg/kg) and PBD drug exposure (μg/m²);and placebo buffer control (Vehicle) and monitored over two weeks ormore to measure the time to tumor doubling, log cell kill, and tumorshrinkage.

Use

The conjugates of the invention may be used to provide a PBD compound ata target location.

The target location is preferably a proliferative cell population. Theantibody is an antibody for an antigen present on a proliferative cellpopulation.

In one embodiment the antigen is absent or present at a reduced level ina non-proliferative cell population compared to the amount of antigenpresent in the proliferative cell population, for example a tumour cellpopulation.

At the target location the linker may be cleaved so as to release acompound RelA or RelB.

Thus, the conjugate may be used to selectively provide a compound RelAor RelB to the target location.

The linker may be cleaved by an enzyme present at the target location.

The target location may be in vitro, in vivo or ex vivo.

The antibody-drug conjugate (ADC) compounds of the invention includethose with utility for anticancer activity. In particular, the compoundsinclude an antibody conjugated, i.e. covalently attached by a linker, toa PBD drug moiety, i.e. toxin. When the drug is not conjugated to anantibody, the PBD drug has a cytotoxic effect. The biological activityof the PBD drug moiety is thus modulated by conjugation to an antibody.The antibody-drug conjugates (ADC) of the invention selectively deliveran effective dose of a cytotoxic agent to tumor tissue whereby greaterselectivity, i.e. a lower efficacious dose, may be achieved.

Thus, in one aspect, the present invention provides a conjugate compoundas described herein for use in therapy.

In a further aspect there is also provides a conjugate compound asdescribed herein for use in the treatment of a proliferative disease. Asecond aspect of the present invention provides the use of a conjugatecompound in the manufacture of a medicament for treating a proliferativedisease.

One of ordinary skill in the art is readily able to determine whether ornot a candidate conjugate treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

The term “proliferative disease” pertains to an unwanted or uncontrolledcellular proliferation of excessive or abnormal cells which isundesired, such as, neoplastic or hyperplastic growth, whether in vitroor in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g. histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), lymphomas, leukemias,psoriasis, bone diseases, fibroproliferative disorders (e.g. ofconnective tissues), and atherosclerosis. Cancers of particular interestinclude, but are not limited to, leukemias and ovarian cancers.

Any type of cell may be treated, including but not limited to, lung,gastrointestinal (including, e.g. bowel, colon), breast (mammary),ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas,brain, and skin.

In one embodiment, the treatment is of a pancreatic cancer.

In one embodiment, the treatment is of a tumour having α_(v)β₆ integrinon the surface of the cell.

It is contemplated that the antibody-drug conjugates (ADC) of thepresent invention may be used to treat various diseases or disorders,e.g. characterized by the overexpression of a tumor antigen. Exemplaryconditions or hyperproliferative disorders include benign or malignanttumors; leukemia, haematological, and lymphoid malignancies. Othersinclude neuronal, glial, astrocytal, hypothalamic, glandular,macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenicand immunologic, including autoimmune, disorders.

Generally, the disease or disorder to be treated is a hyperproliferativedisease such as cancer. Examples of cancer to be treated herein include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g. epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

Autoimmune diseases for which the ADC compounds may be used in treatmentinclude rheumatologic disorders (such as, for example, rheumatoidarthritis, Sjögren's syndrome, scleroderma, lupus such as SLE and lupusnephritis, polymyositis/dermatomyositis, cryoglobulinemia,anti-phospholipid antibody syndrome, and psoriatic arthritis),osteoarthritis, autoimmune gastrointestinal and liver disorders (suchas, for example, inflammatory bowel diseases (e.g. ulcerative colitisand Crohn's disease), autoimmune gastritis and pernicious anemia,autoimmune hepatitis, primary biliary cirrhosis, primary sclerosingcholangitis, and celiac disease), vasculitis (such as, for example,ANCA-associated vasculitis, including Churg-Strauss vasculitis,Wegener's granulomatosis, and polyarteriitis), autoimmune neurologicaldisorders (such as, for example, multiple sclerosis, opsoclonusmyoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson'sdisease, Alzheimer's disease, and autoimmune polyneuropathies), renaldisorders (such as, for example, glomerulonephritis, Goodpasture'ssyndrome, and Berger's disease), autoimmune dermatologic disorders (suchas, for example, psoriasis, urticaria, hives, pemphigus vulgaris,bullous pemphigoid, and cutaneous lupus erythematosus), hematologicdisorders (such as, for example, thrombocytopenic purpura, thromboticthrombocytopenic purpura, post-transfusion purpura, and autoimmunehemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases(such as, for example, inner ear disease and hearing loss), Behcet'sdisease, Raynaud's syndrome, organ transplant, and autoimmune endocrinedisorders (such as, for example, diabetic-related autoimmune diseasessuch as insulin-dependent diabetes mellitus (IDDM), Addison's disease,and autoimmune thyroid disease (e.g. Graves' disease and thyroiditis)).More preferred such diseases include, for example, rheumatoid arthritis,ulcerative colitis, ANCA-associated vasculitis, lupus, multiplesclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

Methods of Treatment

The conjugates of the present invention may be used in a method oftherapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a conjugate compound of theinvention. The term “therapeutically effective amount” is an amountsufficient to show benefit to a patient. Such benefit may be at leastamelioration of at least one symptom. The actual amount administered,and rate and time-course of administration, will depend on the natureand severity of what is being treated. Prescription of treatment, e.g.decisions on dosage, is within the responsibility of generalpractitioners and other medical doctors.

A compound of the invention may be administered alone or in combinationwith other treatments, either simultaneously or sequentially dependentupon the condition to be treated. Examples of treatments and therapiesinclude, but are not limited to, chemotherapy (the administration ofactive agents, including, e.g. drugs, such as chemotherapeutics);surgery; and radiation therapy.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.

Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum (II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®,Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, II),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, calicheamicin gamma1l, calicheamicin omegal1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rlL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),ofatumumab (ARZERRA®, GSK), pertuzumab (PERJETA™ OMNITARG™, 2C4,Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar,Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin(MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the conjugates of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a conjugate compound, a pharmaceuticallyacceptable excipient, carrier, buffer, stabiliser or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The precise nature of the carrier or other material willdepend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Formulations

While it is possible for the conjugate compound to be used (e.g.,administered) alone, it is often preferable to present it as acomposition or formulation.

In one embodiment, the composition is a pharmaceutical composition(e.g., formulation, preparation, medicament) comprising a conjugatecompound, as described herein, and a pharmaceutically acceptablecarrier, diluent, or excipient.

In one embodiment, the composition is a pharmaceutical compositioncomprising at least one conjugate compound, as described herein,together with one or more other pharmaceutically acceptable ingredientswell known to those skilled in the art, including, but not limited to,pharmaceutically acceptable carriers, diluents, excipients, adjuvants,fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers,solubilisers, surfactants (e.g., wetting agents), masking agents,colouring agents, flavouring agents, and sweetening agents.

In one embodiment, the composition further comprises other activeagents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, Handbook of PharmaceuticalAdditives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (SynapseInformation Resources, Inc., Endicott, N.Y., USA), Remington'sPharmaceutical Sciences, 20th edition, pub. Lippincott, Williams &Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition,1994.

Another aspect of the present invention pertains to methods of making apharmaceutical composition comprising admixing at least one[¹¹C]-radiolabelled conjugate or conjugate-like compound, as definedherein, together with one or more other pharmaceutically acceptableingredients well known to those skilled in the art, e.g., carriers,diluents, excipients, etc. If formulated as discrete units (e.g.,tablets, etc.), each unit contains a predetermined amount (dosage) ofthe active compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive compound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with carriers(e.g., liquid carriers, finely divided solid carrier, etc.), and thenshaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the activeingredient is dissolved, suspended, or otherwise provided (e.g., in aliposome or other microparticulate). Such liquids may additional containother pharmaceutically acceptable ingredients, such as anti-oxidants,buffers, preservatives, stabilisers, bacteriostats, suspending agents,thickening agents, and solutes which render the formulation isotonicwith the blood (or other relevant bodily fluid) of the intendedrecipient. Examples of excipients include, for example, water, alcohols,polyols, glycerol, vegetable oils, and the like. Examples of suitableisotonic carriers for use in such formulations include Sodium ChlorideInjection, Ringer's Solution, or Lactated Ringer's Injection. Typically,the concentration of the active ingredient in the liquid is from about 1ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1μg/ml. The formulations may be presented in unit-dose or multi-dosesealed containers, for example, ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the conjugate compound, and compositions comprising theconjugate compound, can vary from patient to patient. Determining theoptimal dosage will generally involve the balancing of the level oftherapeutic benefit against any risk or deleterious side effects. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular compound, the route ofadministration, the time of administration, the rate of excretion of thecompound, the duration of the treatment, other drugs, compounds, and/ormaterials used in combination, the severity of the condition, and thespecies, sex, age, weight, condition, general health, and prior medicalhistory of the patient. The amount of compound and route ofadministration will ultimately be at the discretion of the physician,veterinarian, or clinician, although generally the dosage will beselected to achieve local concentrations at the site of action whichachieve the desired effect without causing substantial harmful ordeleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the active compound is in the range ofabout 100 ng to about 25 mg (more typically about 1 μg to about 10 mg)per kilogram body weight of the subject per day. Where the activecompound is a salt, an ester, an amide, a prodrug, or the like, theamount administered is calculated on the basis of the parent compoundand so the actual weight to be used is increased proportionately.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 100 mg, 3 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 150 mg, 2 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 200 mg, 2 timesdaily.

However in one embodiment, the conjugate compound is administered to ahuman patient according to the following dosage regime: about 50 orabout 75 mg, 3 or 4 times daily.

In one embodiment, the conjugate compound is administered to a humanpatient according to the following dosage regime: about 100 or about 125mg, 2 times daily.

The dosage amounts described above may apply to the conjugate (includingthe PBD moiety and the linker to the antibody) or to the effectiveamount of PBD compound provided, for example the amount of compound thatis releasable after cleavage of the linker.

For the prevention or treatment of disease, the appropriate dosage of anADC of the invention will depend on the type of disease to be treated,as defined above, the severity and course of the disease, whether themolecule is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The molecule issuitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. An exemplarydosage of ADC to be administered to a patient is in the range of about0.1 to about 10 mg/kg of patient weight. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs. Anexemplary dosing regimen comprises a course of administering an initialloading dose of about 4 mg/kg, followed by additional doses every week,two weeks, or three weeks of an ADC. Other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques and assays.

Treatment

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, regression of the condition,amelioration of the condition, and cure of the condition. Treatment as aprophylactic measure (i.e., prophylaxis, prevention) is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosagefrom comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio, when administered in accordance with a desiredtreatment regimen.

Similarly, the term “prophylactically-effective amount,” as used herein,pertains to that amount of an active compound, or a material,composition or dosage from comprising an active compound, which iseffective for producing some desired prophylactic effect, commensuratewith a reasonable benefit/risk ratio, when administered in accordancewith a desired treatment regimen.

Preparation of Drug Conjugates

Antibody drug conjugates, as well as conjugates with other cell bindingagents, may be prepared by several routes, employing organic chemistryreactions, conditions, and reagents known to those skilled in the art,including reaction of a nucleophilic group of an antibody or cellbinding agent with a drug-linker reagent. This method may be employedwith a variety of antibodies and cell binding agents to prepare theantibody-drug conjugates of the invention.

Nucleophilic groups on antibodies include, but are not limited to sidechain thiol groups, e.g. cysteine. Thiol groups are nucleophilic andcapable of reacting to form covalent bonds with electrophilic groups onlinker moieties such as those of the present invention. Certainantibodies have reducible interchain disulfides, i.e. cysteine bridges.Antibodies may be made reactive for conjugation with linker reagents bytreatment with a reducing agent such as DTT (Cleland's reagent,dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride;Getz et al (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures,Beverly, Mass.). Each cysteine disulfide bridge will thus form,theoretically, two reactive thiol nucleophiles. Additional nucleophilicgroups can be introduced into antibodies through the reaction of lysineswith 2-iminothiolane (Traut's reagent) resulting in conversion of anamine into a thiol.

The Subject/Patient

The subject/patient may be an animal, mammal, a placental mammal, amarsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilledplatypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse),murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., abird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., ahorse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., acow), a primate, simian (e.g., a monkey or ape), a monkey (e.g.,marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang,gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,for example, a foetus. In one preferred embodiment, the subject/patientis a human.

In one embodiment, the patient is a population where each patient has atumour having a_(v)46 integrin on the surface of the cell.

EXAMPLES General Experimental Methods for Examples 1 and 2

Optical rotations were measured on an ADP 220 polarimeter (BellinghamStanley Ltd.) and concentrations (c) are given in g/100 mL. Meltingpoints were measured using a digital melting point apparatus(Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum1000 FT IR Spectrometer. ¹H and ¹³C NMR spectra were acquired at 300 Kusing a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively.Chemical shifts are reported relative to TMS (6=0.0 ppm), and signalsare designated as s (singlet), d (doublet), t (triplet), dt (doubletriplet), dd (doublet of doublets), ddd (double doublet of doublets) orm (multiplet), with coupling constants given in Hertz (Hz). Massspectroscopy (MS) data were collected using a Waters Micromass ZQinstrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. WatersMicromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35;Extractor (V), 3.0; Source temperature (° C.), 100; DesolvationTemperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flowrate (L/h), 250. High-resolution mass spectroscopy (HRMS) data wererecorded on a Waters Micromass QTOF Global in positive W-mode usingmetal-coated borosilicate glass tips to introduce the samples into theinstrument. Thin Layer Chromatography (TLC) was performed on silica gelaluminium plates (Merck 60, F₂₅₄), and flash chromatography utilisedsilica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt(NovaBiochem) and solid-supported reagents (Argonaut), all otherchemicals and solvents were purchased from Sigma-Aldrich and were usedas supplied without further purification. Anhydrous solvents wereprepared by distillation under a dry nitrogen atmosphere in the presenceof an appropriate drying agent, and were stored over 4 Å molecularsieves or sodium wire. Petroleum ether refers to the fraction boiling at40-60° C.

General LC/MS conditions: The HPLC (Waters Alliance 2695) was run usinga mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B)(formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then5% B to 95% B over 2.5 min. The composition was held for 0.5 min at 95%B, and then returned to 5% B in 0.1 minutes and held there for 0.9 min.Total gradient run time equals 5 min. Flow rate 3.0 mL/min, 400 μL wassplit via a zero dead volume tee piece which passes into the massspectrometer. Wavelength detection range: 220 to 400 nm. Function type:diode array (535 scans). Column: Phenomenex® Onyx Monolithic C18 50×4.60mm

Example 1 (i) (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)methanone(9)

(a) 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzaldehyde (2)

Neat triisopropylsilylchloride (56.4 mL, 262 mmol) was added to amixture of imidazole (48.7 g, 715.23 mmol) and4-hydroxy-5-methoxy-2-nitrobenzaldehyde 1 (47 g, 238 mmol) (groundtogether). The mixture was heated until the phenol and imidazole meltedand went into solution (100° C.). The reaction mixture was allowed tostir for 15 minutes and was then allowed to cool, whereupon a solid wasobserved to form at the bottom of the flask (imidazole chloride). Thereaction mixture was diluted with 5% EtOAc/hexanes and loaded directlyonto silica gel and the pad was eluted with 5% EtOAc/hexanes, followedby 10% EtOAc/hexanes (due to the low excess, very little unreactedTIPSCI was found in the product). The desired product was eluted with 5%ethyl acetate in hexane. Excess eluent was removed by rotary evaporationunder reduced pressure, followed by drying under high vacuum to afford acrystalline light sensitive solid (74.4 g, 88%). Purity satisfactory byLC/MS (4.22 min (ES+) m/z (relative intensity) 353.88 ([M+H]⁺, 100)); ¹HNMR (400 MHz, CDCl₃) δ 10.43 (s, 1H), 7.60 (s, 1H), 7.40 (s, 1H), 3.96(s, 3H), 1.35-1.24 (m, 3H), 1.10 (m, 18H).

(b) 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid (3)

A solution of sodium chlorite (47.3 g, 523 mmol, 80% technical grade)and sodium dihydrogenphosphate monobasic (35.2 g, 293 mmol) (NaH₂PO₄) inwater (800 mL) was added to a solution of compound 2 (74 g, 209 mmol) intetrahydrofuran (500 mL) at room temperature. Hydrogen peroxide (60%w/w, 140 mL, 2.93 mol) was immediately added to the vigorously stirredbiphasic mixture. The reaction mixture evolved gas (oxygen), thestarting material dissolved and the temperature of the reaction mixturerose to 45° C. After 30 minutes LC/MS revealed that the reaction wascomplete. The reaction mixture was cooled in an ice bath andhydrochloric acid (1 M) was added to lower the pH to 3 (this step wasfound unnecessary in many instances, as the pH at the end of thereaction is already acidic; please check the pH before extraction). Thereaction mixture was then extracted with ethyl acetate (1 L) and theorganic phases washed with brine (2×100 mL) and dried over magnesiumsulphate. The organic phase was filtered and excess solvent removed byrotary evaporation under reduced pressure to afford the product 3 inquantitative yield as a yellow solid. LC/MS (3.93 min (ES−) m/z(relative intensity) 367.74 ([M−H]⁻, 100)); ¹H NMR (400 MHz, CDCl₃) δ7.36 (s, 1H), 7.24 (s, 1H), 3.93 (s, 3H), 1.34-1.22 (m, 3H), 1.10 (m,18H).

(c)((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (5)

DCC (29.2 g, 141 mmol, 1.2 eq) was added to a solution of acid 3 (43.5g, 117.8 mmol, leq), and hydroxybenzotriazole hydrate (19.8 g, 129.6mmol, 1.1 eq) in dichloromethane (200 mL) at 0° C. The cold bath wasremoved and the reaction was allowed to proceed for 30 mins at roomtemperature, at which time a solution of(2S,4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine 4 (30 g,129.6 mmol, 1.1 eq) and triethylamine (24.66 mL, 176 mmol, 1.5 eq) indichloromethane (100 mL) was added rapidly at −10° C. under argon (onlarge scale, the addition time could be shortened by cooling thereaction mixture even further. The reaction mixture was allowed to stirat room temperature for 40 minutes to 1 hour and monitored by LC/MS andTLC (EtOAc). The solids were removed by filtration over celite and theorganic phase was washed with cold aqueous 0.1 M HCl until the pH wasmeasured at 4 or 5. The organic phase was then washed with water,followed by saturated aqueous sodium bicarbonate and brine. The organiclayer was dried over magnesium sulphate, filtered and excess solventremoved by rotary evaporation under reduced pressure. The residue wassubjected to column flash chromatography (silica gel; gradient 40/60ethyl acetate/hexane to 80/20 ethyl acetate/hexane). Excess solvent wasremoved by rotary evaporation under reduced pressure afforded the pureproduct 5, (45.5 g of pure product 66%, and 17 g of slightly impureproduct, 90% in total). LC/MS 4.43 min (ES+) m/z (relative intensity)582.92 ([M+H]⁺, 100); ¹H NMR (400 MHz, CDCl₃) δ 7.66 (s, 1H), 6.74 (s,1H), 4.54 (s, 1H), 4.40 (s, 1H), 4.13 (s, 1H), 3.86 (s, 3H), 3.77 (d,J=9.2 Hz, 1H), 3.36 (dd, J=11.3, 4.5 Hz, 1H), 3.14-3.02 (m, 1H),2.38-2.28 (m, 1H), 2.10 (ddd, J=13.3, 8.4, 2.2 Hz, 1H), 1.36-1.19 (m,3H), 1.15-1.05 (m, 18H), 0.91 (s, 9H), 0.17-0.05 (m, 6H), (presence ofrotamers).

(d)(S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)pyrrolidin-3-one (6)

TCCA (8.82 g, 40 mmol, 0.7 eq) was added to a stirred solution of 5(31.7 g, 54 mmol, 1 eq) and TEMPO (0.85 g, 5.4 mmol, 0.1 eq) in drydichloromethane (250 mL) at 0° C. The reaction mixture was vigorouslystirred for 20 minutes, at which point TLC (50/50 ethyl acetate/hexane)revealed complete consumption of the starting material. The reactionmixture was filtered through celite and the filtrate washed with aqueoussaturated sodium bicarbonate (100 mL), sodium thiosulphate (9 g in 300mL), brine (100 mL) and dried over magnesium sulphate. Rotaryevaporation under reduced pressure afforded product 6 in quantitativeyield. LC/MS 4.52 min (ES+) m/z (relative intensity) 581.08 ([M+H]⁺,100);

¹H NMR (400 MHz, CDCl₃) δ 7.78-7.60 (m, 1H), 6.85-6.62 (m, 1H), 4.94(dd, J=30.8, 7.8 Hz, 1H), 4.50-4.16 (m, 1H), 3.99-3.82 (m, 3H),3.80-3.34 (m, 3H), 2.92-2.17 (m, 2H), 1.40-1.18 (m, 3H), 1.11 (t, J=6.2Hz, 18H), 0.97-0.75 (m, 9H), 0.15-1-0.06 (m, 6H), (presence ofrotamers).

(e)(S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)-4,5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate (7)

Triflic anhydride (27.7 mL, 46.4 g, 165 mmol, 3 eq) was injected(temperature controlled) to a vigorously stirred suspension of ketone 6(31.9 g, 55 mmol, 1 eq) in dry dichloromethane (900 mL) in the presenceof 2,6-lutidine (25.6 mL, 23.5 g, 220 mmol, 4 eq, dried over sieves) at−50° C. (acetone/dry ice bath). The reaction mixture was allowed to stirfor 1.5 hours when LC/MS, following a mini work-up(water/dichloromethane), revealed the reaction to be complete. Water wasadded to the still cold reaction mixture and the organic layer wasseparated and washed with saturated sodium bicarbonate, brine andmagnesium sulphate. The organic phase was filtered and excess solventwas removed by rotary evaporation under reduced pressure. The residuewas subjected to column flash chromatography (silica gel; 10/90 v/vethyl acetate/hexane), removal of excess eluent afforded the product 7(37.6 g, 96%) LC/MS, method 2, 4.32 min (ES+) m/z (relative intensity)712.89 ([M+H]⁺, 100); ¹H NMR (400 MHz, CDCl₃) δ 7.71 (s, 1H), 6.75 (s,1H), 6.05 (d, J=1.8 Hz, 1H), 4.78 (dd, J=9.8, 5.5 Hz, 1H), 4.15-3.75 (m,5H), 3.17 (ddd, J=16.2, 10.4, 2.3 Hz, 1H), 2.99 (ddd, J=16.3, 4.0, 1.6Hz, 1H), 1.45-1.19 (m, 3H), 1.15-1.08 (m, 18H), 1.05 (s, 6H), 0.95-0.87(m, 9H), 0.15-0.08 (m, 6H).

(f)(S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone(8)

Triphenylarsine (1.71 g, 5.60 mmol, 0.4 eq) was added to a mixture oftriflate 7 (10.00 g, 14 mmol, 1 eq), methylboronic acid (2.94 g, 49.1mmol, 3.5 eq), silver oxide (13 g, 56 mmol, 4 eq) and potassiumphosphate tribasic (17.8 g, 84 mmol, 6 eq) in dry dioxane (80 mL) underan argon atmosphere. The reaction was flushed with argon 3 times andbis(benzonitrile)palladium(II) chloride (540 mg, 1.40 mmol, 0.1 eq) wasadded. The reaction was flushed with argon 3 more times before beingwarmed instantaneously to 110° C. (the drysyn heating block waspreviously warmed to 110° C. prior addition of the flask). After 10 minsthe reaction was cooled to room temperature and filtered through a padcelite. The solvent was removed by rotary evaporation under reducedpressure. The resulting residue was subjected to column flashchromatography (silica gel; 10% ethyl acetate/hexane). Pure fractionswere collected and combined, and excess eluent was removed by rotaryevaporation under reduced pressure afforded the product 8 (4.5 g, 55%).LC/MS, 4.27 min (ES+) m/z (relative intensity) 579.18 ([M+H]⁺, 100); ¹HNMR (400 MHz, CDCl₃) δ 7.70 (s, 1H), 6.77 (s, 1H), 5.51 (d, J=1.7 Hz,1H), 4.77-4.59 (m, 1H), 3.89 (s, 3H), 2.92-2.65 (m, 1H), 2.55 (d, J=14.8Hz, 1H), 1.62 (d, J=1.1 Hz, 3H), 1.40-1.18 (m, 3H), 1.11 (s, 9H), 1.10(s, 9H), 0.90 (s, 9H), 0.11 (d, J=2.3 Hz, 6H).

(g) (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)methanone(9)

Zinc powder (28 g, 430 mmol, 37 eq) was added to a solution of compound8 (6.7 g, 11.58 mmol) in 5% formic acid in ethanol v/v (70 mL) at around15° C. The resulting exotherm was controlled using an ice bath tomaintain the temperature of the reaction mixture below 30° C. After 30minutes the reaction mixture was filtered through a pad of celite. Thefiltrate was diluted with ethyl acetate and the organic phase was washedwith water, saturated aqueous sodium bicarbonate and brine. The organicphase was dried over magnesium sulphate, filtered and excess solventremoved by rotary evaporation under reduced pressure. The resultingresidue was subjected to flash column chromatography (silica gel; 10%ethyl acetate in hexane). The pure fractions were collected and combinedand excess solvent was removed by rotary evaporation under reducedpressure to afford the product 9 (5.1 g, 80%). LC/MS, 4.23 min (ES+) m/z(relative intensity) 550.21 ([M+H]⁺, 100); ¹H NMR (400 MHz, CDCl₃) δ7.28 (s, 1H), 6.67 (s, 1H), 6.19 (s, 1H), 4.64-4.53 (m, J=4.1 Hz, 1H),4.17 (s, 1H), 3.87 (s, 1H), 3.77-3.69 (m, 1H), 3.66 (s, 3H), 2.71-2.60(m, 1H), 2.53-2.43 (m, 1H), 2.04-1.97 (m, J=11.9 Hz, 1H), 1.62 (s, 3H),1.26-1.13 (m, 3H), 1.08-0.99 (m, 18H), 0.82 (s, 9H), 0.03-1-0.03 (m,J=6.2 Hz, 6H).

(ii) (11 S, 11 aS)-allyl11-((tert-butyldimethylsilyl)oxy)-8-((5-iodopentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate

(a) (S)-allyl (2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate(10)

Allyl chloroformate (0.30 mL, 3.00 mmol, 1.1 eq) was added to a solutionof amine 9 (1.5 g, 2.73 mmol) in the presence of dry pyridine (0.48 mL,6.00 mmol, 2.2 eq) in dry dichloromethane (20 mL) at −78° C.(acetone/dry ice bath). After 30 minutes, the bath was removed and thereaction mixture was allowed to warm to room temperature. The reactionmixture was diluted with dichloromethane and saturated aqueous coppersulphate was added. The organic layer was then washed sequentially withsaturated aqueous sodium bicarbonate and brine. The organic phase wasdried over magnesium sulphate, filtered and excess solvent removed byrotary evaporation under reduced pressure to afford the product 10 whichwas used directly in the next reaction. LC/MS, 4.45 min (ES+) m/z(relative intensity) 632.91 ([M+H]⁺, 100)

(b) (S)-allyl(2-(2-(hydroxymethyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (11)

The crude 10 was dissolved in a 7:1:1:2 mixture of aceticacid/methanol/tetrahydrofuran/water (28:4:4:8 mL) and allowed to stir atroom temperature. After 3 hours, complete disappearance of startingmaterial was observed by LC/MS. The reaction mixture was diluted withethyl acetate and washed sequentially with water (2×500 mL), saturatedaqueous sodium bicarbonate (200 mL) and brine. The organic phase wasdried over magnesium sulphate filtered and excess ethyl acetate removedby rotary evaporation under reduced pressure. The resulting residue wassubjected to flash column chromatography (silica gel, 25% ethyl acetatein hexane). Pure fractions were collected and combined and excess eluentwas removed by rotary evaporation under reduced pressure to afford thedesired product 11 (1 g, 71%). LC/MS, 3.70 min (ES+) m/z (relativeintensity) 519.13 ([M+H]⁺, 95); ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H),7.69 (s, 1H), 6.78 (s, 1H), 6.15 (s, 1H), 5.95 (ddt, J=17.2, 10.5, 5.7Hz, 1H), 5.33 (dq, J=17.2, 1.5 Hz, 1H), 5.23 (ddd, J=10.4, 2.6, 1.3 Hz,1H), 4.73 (tt, J=7.8, 4.8 Hz, 1H), 4.63 (dt, J=5.7, 1.4 Hz, 2H), 4.54(s, 1H), 3.89-3.70 (m, 5H), 2.87 (dd, J=16.5, 10.5 Hz, 1H), 2.19 (dd,J=16.8, 4.6 Hz, 1H), 1.70 (d, J=1.3 Hz, 3H), 1.38-1.23 (m, 3H), 1.12 (s,10H), 1.10 (s, 8H).

(c) (11 S, 11aS)-allyl11-hydroxy-7-methoxy-2-methyl-5-oxo-8-((triisopropylslyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][,4]diazepine-10(5H)-carboxylate (12)

Dimethyl sulphoxide (0.35 mL, 4.83 mmol, 2.5 eq) was added dropwise to asolution of oxalyl chloride (0.2 mL, 2.32 mmol, 1.2 eq) in drydichloromethane (10 mL) at −78° C. (dry ice/acetone bath) under anatmosphere of argon. After 10 minutes a solution of 11 (1 g, 1.93 mmol)in dry dichloromethane (8 mL) was added slowly with the temperaturestill at −78° C. After 15 min triethylamine (1.35 mL, dried over 4 Åmolecular sieves, 9.65 mmol, 5 eq) was added dropwise and the dryice/acetone bath was removed. The reaction mixture was allowed to reachroom temperature and was extracted with cold hydrochloric acid (0.1 M),saturated aqueous sodium bicarbonate and brine. The organic phase wasdried over magnesium sulphate, filtered and excess dichloromethane wasremoved by rotary evaporation under reduced pressure to afford product12 (658 mg, 66%). LC/MS, 3.52 min (ES+) m/z (relative intensity) 517.14([M+H]⁺, 100); ¹H NMR (400 MHz, CDCl₃) δ 7.20 (s, 1H), 6.75-6.63 (m,J=8.8, 4.0 Hz, 2H), 5.89-5.64 (m, J=9.6, 4.1 Hz, 2H), 5.23-5.03 (m, 2H),4.68-4.38 (m, 2H), 3.84 (s, 3H), 3.83-3.77 (m, 1H), 3.40 (s, 1H),3.05-2.83 (m, 1H), 2.59 (d, J=17.1 Hz, 1H), 1.78 (d, J=1.3 Hz, 3H),1.33-1.16 (m, 3H), 1.09 (d, J=2.2 Hz, 9H), 1.07 (d, J=2.1 Hz, 9H).

(d) (11S,11aS)-allyl11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(13)

Tert-butyldimethylsilyltriflate (0.70 mL, 3.00 mmol, 3 eq) was added toa solution of compound 12 (520 mg, 1.00 mmol) and 2,6-lutidine (0.46 mL,4.00 mmol, 4 eq) in dry dichloromethane (40 mL) at 0° C. under argon.After 10 min, the cold bath was removed and the reaction mixture wasstirred at room temperature for 1 hour. The reaction mixture wasextracted with water, saturated aqueous sodium bicarbonate and brine.The organic phase was dried over magnesium sulphate, filtered and excesswas removed by rotary evaporation under reduced pressure. The resultingresidue was subjected to flash column chromatography (silica gel;gradient, 10% ethyl acetate in hexane to 20% ethyl acetate in hexane).Pure fractions were collected and combined and excess eluent was removedby rotary evaporation under reduced pressure to give the product 13 (540mg, 85%). LC/MS, 4.42 min (ES+) m/z (relative intensity) 653.14([M+Na]⁺, 100); ¹H NMR (400 MHz, CDCl₃) δ 7.20 (s, 1H), 6.71-6.64 (m,J=5.5 Hz, 2H), 5.83 (d, J=9.0 Hz, 1H), 5.80-5.68 (m, J=5.9 Hz, 1H),5.14-5.06 (m, 2H), 4.58 (dd, J=13.2, 5.2 Hz, 1H), 4.36 (dd, J=13.3, 5.5Hz, 1H), 3.84 (s, 3H), 3.71 (td, J=10.1, 3.8 Hz, 1H), 2.91 (dd, J=16.9,10.3 Hz, 1H), 2.36 (d, J=16.8 Hz, 1H), 1.75 (s, 3H), 1.31-1.16 (m, 3H),1.12-1.01 (m, J=7.4, 2.1 Hz, 18H), 0.89-0.81 (m, 9H), 0.25 (s, 3H), 0.19(s, 3H).

(e) (11S,11aS)-allyl11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(14)

Lithium acetate (87 mg, 0.85 mmol) was added to a solution of compound13 (540 mg, 0.85 mmol) in wet dimethylformamide (6 mL, 50:1 DMF/water).After 4 hours, the reaction was complete and the reaction mixture wasdiluted with ethyl acetate (25 mL) and washed with aqueous citric acidsolution (pH ˜3), water and brine. The organic layer was dried overmagnesium sulphate filtered and excess ethyl acetate was removed byrotary evaporation under reduced pressure. The resulting residue wassubjected to flash column chromatography (silica gel; gradient, 25% to75% ethyl acetate in hexane). Pure fractions were collected and combinedand excess eluent was removed by rotary evaporation under reducedpressure to give the product 14 (400 mg, quantitative). LC/MS, (3.33 min(ES+) m/z (relative intensity) 475.26 ([M+H]⁺, 100).

(f)(11S,11aS)-allyl11-((tert-butyldimethylsilyl)oxy)-8-((5-iodopentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(15)

Diiodopentane (0.63 mL, 4.21 mmol, 5 eq) and potassium carbonate (116mg, 0.84 mmol, 1 eq) were added to a solution of phenol 14 (400 mg, 0.84mmol) in acetone (4 mL, dried over molecular sieves). The reactionmixture was then warmed to 60° C. and stirred for 6 hours. Acetone wasremoved by rotary evaporation under reduced pressure. The resultingresidue was subjected to flash column chromatography (silica gel; 50/50,v/v, hexane/ethyl acetate). Pure fractions were collected and combinedand excess eluent was removed to provide 15 in 90% yield. LC/MS, 3.90min (ES+) m/z (relative intensity) 670.91 ([M]⁺, 100). ¹H NMR (400 MHz,CDCl₃) δ 7.23 (s, 1H), 6.69 (s, 1H), 6.60 (s, 1H), 5.87 (d, J=8.8 Hz,1H), 5.83-5.68 (m, J=5.6 Hz, 1H), 5.15-5.01 (m, 2H), 4.67-4.58 (m, 1H),4.45-4.35 (m, 1H), 4.04-3.93 (m, 2H), 3.91 (s, 3H), 3.73 (td, J=10.0,3.8 Hz, 1H), 3.25-3.14 (m, J=8.5, 7.0 Hz, 2H), 2.92 (dd, J=16.8, 10.3Hz, 1H), 2.38 (d, J=16.8 Hz, 1H), 1.95-1.81 (m, 4H), 1.77 (s, 3H),1.64-1.49 (m, 2H), 0.88 (s, 9H), 0.25 (s, 3H), 0.23 (s, 3H).

(iii)(11S,11aS)-4-(2-(1-((1-(allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(20)

(a) Allyl3-(2-(2-(4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamoyl)oxy) methyl)phenyl)hydrazinyl)propanamido)-4-methyl-2-oxopentanoate (16)

Triethylamine (2.23 mL, 18.04 mmol, 2.2 eq) was added to a stirredsolution of the amine 9 (4 g, 8.20 mmol) and triphosgene (778 mg, 2.95mmol, 0.36 eq) in dry tetrahydrofuran (40 mL) at 5° C. (ice bath). Theprogress of the isocyanate reaction was monitored by periodicallyremoving aliquots from the reaction mixture and quenching with methanoland performing LC/MS analysis. Once the isocyanate formation wascomplete a solution of the alloc-Val-Ala-PABOH (4.12 g, 12.30 mmol, 1.5eq) and triethylamine (1.52 mL, 12.30 mmol, 1.5 eq) in drytetrahydrofuran (40 mL) was rapidly added by injection to the freshlyprepared isocyanate. The reaction mixture was allowed to stir at 40° C.for 4 hours. Excess solvent was removed by rotary evaporation underreduced pressure. The resulting residue was subjected to flash columnchromatography (silica gel; gradient, 1% methanol to 5% methanol indichloromethane). (Alternative chromatography conditions using EtOAc andHexane have also been successful). Pure fractions were collected andcombined and excess eluent was removed by rotary evaporation underreduced pressure to give the 5 product 16 (3.9 g, 50%). LC/MS, 4.23 min(ES+) m/z (relative intensity) 952.36 ([M+H]⁺, 100); ¹H NMR (400 MHz,CDCl₃) δ 8.62 (br s, 1H), 8.46 (s, 1H), 7.77 (br s, 1H), 7.53 (d, J=8.4Hz, 2H), 7.32 (d, J=8.5 Hz, 2H), 6.76 (s, 1H), 6.57 (d, J=7.6 Hz, 1H),6.17 (s, 1H), 6.03-5.83 (m, 1H), 5.26 (dd, J=33.8, 13.5 Hz, 3H), 5.10(s, 2H), 4.70-4.60 (m, 2H), 4.58 (dd, J=5.7, 1.3 Hz, 2H), 4.06-3.99 (m,1H), 3.92 (s, 1H), 3.82-3.71 (m, 1H), 3.75 (s, 3H), 2.79-2.64 (m, 1H),2.54 (d, J=12.9 Hz, 1H), 2.16 (dq, J=13.5, 6.7 Hz, 1H), 1.67 (s, 3H),1.46 (d, J=7.0 Hz, 3H), 1.35-1.24 (m, 3H), 1.12 (s, 9H), 1.10 (s, 9H),0.97 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz, 3H), 0.87 (s, 9H),0.07-1-0.02 (m, 6H).

(b) Allyl 3-(2-(2-(4-((((2-((S)-2-(hydroxymethyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamoyl)oxy)methyl)phenyl)hydrazinyl)propanamido)-4-methyl-2-oxopentanoate (17)

The TBS ether 16 (1.32 g, 1.38 mmol) was dissolved in a 7:1:1:2 mixtureof acetic acid/methanol/tetrahydrofuran/water (14:2:2:4 mL) and allowedto stir at room temperature. After 3 hours no more starting material wasobserved by LC/MS. The reaction mixture was diluted with ethyl acetate(25 mL) and washed sequentially with water, saturated aqueous sodiumbicarbonate and brine. The organic phase was dried over magnesiumsulphate filtered and excess ethyl acetate removed by rotary evaporationunder reduced pressure. The resulting residue was subjected to flashcolumn chromatography (silica gel, 2% methanol in dichloromethane). Purefractions were collected and combined and excess eluent was removed byrotary evaporation under reduced pressure to afford the desired product17 (920 mg, 80%). LC/MS, 3.60 min (ES+) m/z (relative intensity) 838.18([M+H]⁺, 100). ¹H NMR (400 MHz, CDCl₃) δ 8.55 (s, 1H), 8.35 (s, 1H),7.68 (s, 1H), 7.52 (d, J=8.1 Hz, 2H), 7.31 (d, J=8.4 Hz, 2H), 6.77 (s,1H), 6.71 (d, J=7.5 Hz, 1H), 6.13 (s, 1H), 5.97-5.82 (m, J=5.7 Hz, 1H),5.41-5.15 (m, 3H), 5.10 (d, J=3.5 Hz, 2H), 4.76-4.42 (m, 5H), 4.03 (t,J=6.6 Hz, 1H), 3.77 (s, 5H), 2.84 (dd, J=16.7, 10.4 Hz, 1H), 2.26-2.08(m, 2H), 1.68 (s, 3H), 1.44 (d, J=7.0 Hz, 3H), 1.30 (dt, J=14.7, 7.4 Hz,3H), 1.12 (s, 9H), 1.10 (s, 9H), 0.96 (d, J=6.8 Hz, 3H), 0.93 (d, J=6.8Hz, 3H).

(c) (11S,11aS)-4-(2-(1-((1-(allyloxy)-4-methyl-,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl11-hydroxy-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(18)

Dimethyl sulphoxide (0.2 mL, 2.75 mmol, 2.5 eq) was added dropwise to asolution of oxalyl chloride (0.11 mL, 1.32 mmol, 1.2 eq) in drydichloromethane (7 mL) at −78° C. (dry ice/acetone bath) under anatmosphere of argon. After 10 minutes a solution of 17 (920 mg, 1.10mmol) in dry dichloromethane (5 mL) was added slowly with thetemperature still at −78° C. After 15 min triethylamine (0.77 mL, driedover 4 Å molecular sieves, 5.50 mmol, 5 eq) was added dropwise and thedry ice/acetone bath was removed. The reaction mixture was allowed toreach room temperature and was extracted with cold hydrochloric acid(0.1 M), saturated aqueous sodium bicarbonate and brine. The organicphase was dried over magnesium sulphate, filtered and excessdichloromethane was removed by rotary evaporation under reducedpressure. The resulting residue was subjected to column flashchromatography (silica gel; gradient 2% methanol to 5% methanol indichloromethane). Pure fractions were collected and combined and removalof excess eluent by rotary evaporation under reduced pressure affordedthe product 18 (550 mg, 60%). LC/MS, 3.43 min (ES+) m/z (relativeintensity) 836.01 ([M]⁺, 100). ¹H NMR (400 MHz, CDCl₃) δ 8.39 (s, 1H),7.52-7.40 (m, 2H), 7.21-7.08 (m, J=11.5 Hz, 2H), 6.67 (s, 1H), 6.60-6.47(m, J=7.4 Hz, 1H), 5.97-5.83 (m, 1H), 5.79-5.66 (m, 1H), 5.38-4.90 (m,6H), 4.68-4.52 (m, J=18.4, 5.5 Hz, 4H), 4.04-3.94 (m, J=6.5 Hz, 1H),3.87-3.76 (m, 5H), 3.00-2.88 (m, 1H), 2.66-2.49 (m, 2H), 2.21-2.08 (m,2H), 1.76 (s, 3H), 1.45 (d, J=7.0 Hz, 3H), 1.09-0.98 (m, J=8.9 Hz, 18H),0.96 (d, J=6.7 Hz, 3H), 0.93 (d, J=6.9 Hz, 3H).

(d)(11S,11aS)-4-(2-(1-((1-(Allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(19)

Tert-butyldimethylsilyltriflate (0.38 mL, 1.62 mmol, 3 eq) was added toa solution of compound 18 (450 mg, 0.54 mmol) and 2,6-lutidine (0.25 mL,2.16 mmol, 4 eq) in dry dichloromethane (5 mL) at 0° C. under argon.After 10 min, the cold bath was removed and the reaction mixture wasstirred at room temperature for 1 hour. The reaction mixture wasextracted with water, saturated aqueous sodium bicarbonate and brine.The organic phase was dried over magnesium sulphate, filtered and excesssolvent was removed by rotary evaporation under reduced pressure. Theresulting residue was subjected to column flash chromatography (silicagel; 50/50 v/v hexane/ethyl acetate). Pure fractions were collected andcombined and excess eluent was removed by rotary evaporation underreduced pressure to give the product 19 (334 mg, 65%). LC/MS, 4.18 min(ES+) m/z (relative intensity) 950.50 ([M]⁺, 100). ¹H NMR (400 MHz,CDCl₃) δ 8.53 (s, 1H), 8.02 (s, 1H), 7.44 (d, J=7.6 Hz, 2H), 7.21 (s,1H), 7.08 (d, J=8.2 Hz, 2H), 6.72-6.61 (m, J=8.9 Hz, 2H), 6.16 (s, 1H),5.97-5.79 (m, J=24.4, 7.5 Hz, 2H), 5.41-5.08 (m, 5H), 4.86 (d, J=12.5Hz, 1H), 4.69-4.60 (m, 1H), 4.57 (s, 1H), 4.03 (t, J=6.7 Hz, 1H), 3.87(s, 3H), 3.74 (td, J=9.6, 3.6 Hz, 1H), 2.43-2.09 (m, J=34.8, 19.4, 11.7Hz, 3H), 1.76 (s, 3H), 1.43 (d, J=6.9 Hz, 3H), 1.30-1.21 (m, 3H), 0.97(d, J=6.7 Hz, 3H), 0.92 (t, J=8.4 Hz, 3H), 0.84 (s, 9H), 0.23 (s, 3H),0.12 (s, 3H).

(e)(11S,11aS)-4-(2-(1-((1-(Allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(20)

Lithium acetate (50 mg, 0.49 mmol) was added to a solution of compound19 (470 mg, 0.49 mmol) in wet dimethylformamide (4 mL, 50:1 DMF/water).After 4 hours, the reaction was complete and the reaction mixture wasdiluted with ethyl acetate and washed with citric acid (pH˜3), water andbrine. The organic layer was dried over magnesium sulphate filtered andexcess ethyl acetate was removed by rotary evaporation under reducedpressure. The resulting residue was subjected to column flashchromatography (silica gel; gradient, 50/50 to 25/75 v/v hexane/ethylacetate). Pure fractions were collected and combined and excess eluentwas removed by rotary evaporation under reduced pressure to give theproduct 20 (400 mg, quantitative). LC/MS, 3.32 min (ES+) m/z (relativeintensity) 794.18 ([M+H]⁺, 100). ¹H NMR (400 MHz, CDCl₃) δ 8.53 (s, 1H),8.02 (s, 1H), 7.44 (d, J=7.6 Hz, 2H), 7.21 (s, 1H), 7.08 (d, J=8.2 Hz,2H), 6.72-6.61 (m, J=8.9 Hz, 2H), 6.16 (s, 1H), 5.97-5.79 (m, J=24.4,7.5 Hz, 2H), 5.41-5.08 (m, 5H), 4.86 (d, J=12.5 Hz, 1H), 4.69-4.60 (m,1H), 4.57 (s, 1H), 4.03 (t, J=6.7 Hz, 1H), 3.87 (s, 3H), 3.74 (td,J=9.6, 3.6 Hz, 1H), 2.43-2.09 (m, J=34.8, 19.4, 11.7 Hz, 3H), 1.76 (s,3H), 1.43 (d, J=6.9 Hz, 3H), 1.30-1.21 (m, 3H), 0.97 (d, J=6.7 Hz, 3H),0.92 (t, J=8.4 Hz, 3H), 0.84 (s, 9H), 0.23 (s, 3H), 0.12 (s, 3H).

(iv) (11S,11aS)-4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(24)

(a) (11 S)-allyl8-((5-(((11S)-10-(((4-(2-(1-((1-(allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl)oxy)carbonyl)-1-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(21)

Potassium carbonate (70 mg, 0.504 mmol, 1 eq) was added to a solution of15 (370 mg, 0.552 mmol, 1.2 eq) and phenol 20 (400 mg, 0.504 mmol) indry acetone (25 mL). The reaction was stirred 8 hours at 70° C. TheLC/MS showed that all the starting material was not consumed, so thereaction was allowed to stir overnight at room temperature and stirredfor an additional 2 hours the next day. Acetone was removed by rotaryevaporation under reduced pressure. The resulting residue was subjectedto flash column chromatography (silica gel; 80% ethyl acetate in hexaneto 100% ethyl acetate). Pure fractions were collected and combined andexcess eluent was removed by rotary evaporation under reduced pressureto give the product 21 (385 mg, 57%). LC/MS, 4.07 min (ES+) m/z(relative intensity) 1336.55 ([M+H]⁺, 50).

(b) (11S)-allyl8-((5-(((11S)-10-(((4-(2-(1-((1-(allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(22)

Tetra-n-butylammonium fluoride (1M, 0.34 mL, 0.34 mmol, 2 eq) was addedto a solution of 21 (230 mg, 0.172 mmol) in dry tetrahydrofuran (3 mL).The starting material was totally consumed after 10 minutes. Thereaction mixture was diluted with ethyl acetate (30 mL) and washedsequentially with water and brine. The organic phase was dried overmagnesium sulphate filtered and excess ethyl acetate removed by rotaryevaporation under reduced pressure. The resulting residue 22 was used asa crude mixture for the next reaction. LC/MS, 2.87 min (ES+) m/z(relative intensity) 1108.11 ([M+H]⁺, 100).

(c) (11S)-4-(2-(1-((1-amino-3-methyl-1-oxobutan-2-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl11-hydroxy-7-methoxy-8-((5-((7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(23)

Tetrakis(triphenylphosphine)palladium(0) (12 mg, 0.01 mmol, 0.06 eq) wasadded to a solution of crude 22 (0.172 mmol) and pyrrolidine (36 μL,0.43 mmol, 2.5 eq) in dry dichloromethane (10 mL). The reaction mixturewas stirred 20 minutes and diluted with dichloromethane and washedsequentially with saturated aqueous ammonium chloride and brine. Theorganic phase was dried over magnesium sulphate filtered and excessdichloromethane removed by rotary evaporation under reduced pressure.The resulting residue 23 was used as a crude mixture for the nextreaction. LC/MS, 2.38 min (ES+) m/z (relative intensity) 922.16 ([M+H]⁺;40).

(d)(11S,11aS)-4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(24)

1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDCI, 33 mg, 0.172 mmol)was added to a solution of crude 23 (0.172 mmol) and Mal-(PEG)₈-acid(100 mg, 0.172 mmol) in dry dichloromethane (10 mL). The reaction wasstirred for 2 hours and the presence ofstarting material was no longerobserved by LC/MS. The reaction was diluted with dichloromethane andwashed sequentially with water and brine. The organic phase was driedover magnesium sulphate filtered and excess dichloromethane removed byrotary evaporation under reduced pressure. The resulting residue wassubjected to flash column chromatography (silica gel; 100% chloroform to10% methanol in chloroform). Pure fractions were collected and combinedand excess eluent was removed by rotary evaporation under reducedpressure to give 24 (B) (60 mg, 25% over 3 steps).

Example 2

Compound 25 is compound 79 of WO 2011/130598

(11 S)-4-(1-iodo-20-isopropyl-23-methyl-2, 18,21-trioxo-6,9,12,15-tetraoxa-3,19,22-triazatetracosanamido)benzyl11-hydroxy-7-methoxy-8-(3-((7-methoxy-5-oxo-2-((E)-prop-1-en-1-yl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2-((E)-prop-1-en-1-yl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(26)

N,N′-diisopropylcarbodiimide (DIC, 4.71 μL, 0.0304 mmol) was added to asolution of amine 25 (0.0276 mmol) and Iodo-(PEG)₄-acid (13.1 mg, 0.0304mmol) in dry dichloromethane (0.8 mL). The reaction was stirred for 3hours and the presence of starting material was no longer observed byLC/MS. The reaction mixture was directly loaded onto a thin-layerchromatography (TLC) plate and purified by prep-TLC (10% methanol inchloroform). Pure bands were scraped off the TLC plate, taken up in 10%methanol in chloroform, filtered and excess eluent removed by rotaryevaporation under reduced pressure to give 26 (A) (20.9 mg, 56%). LC/MS,method 2, 3.08 min (ES+) m/z (relative intensity) 1361.16 ([M+H]⁺, 100).

General Experimental Methods for Example 3

LCMS data were obtained using an Agilent 1200 series LC/MS with anAgilent 6110 quadrupole MS, with Electrospray ionisation. Mobile phaseA—0.1% Acetic acid in water. Mobile Phase B—0.1% in acetonitrile. Flowrate of 1.00 ml/min. Gradient from 5% B rising up to 95% B over 3minutes, remaining at 95% B for 1 minute and then back down to 5% B over6 seconds. The total run time is 5 minutes. Column: Phenomenex Gemini-NX3 μm C18, 30×2.00 mm. Chromatograms based on UV detection at 254 nm.Mass Spectra were achieved using the MS in positive mode. Proton NMRchemical shift values were measured on the delta scale at 400 MHz usinga Bruker AV400. The following abbreviations have been used: s, singlet;d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Couplingconstants are reported in Hz. Unless otherwise stated, columnchromatography (by the flash procedure) were performed on MerckKieselgel silica (Art. 9385). Mass spectroscopy (MS) data were collectedusing a Waters Micromass LCT instrument coupled to a Waters 2795 HPLCseparations module. Thin Layer Chromatography (TLC) was performed onsilica gel aluminium plates (Merck 60, F₂₅₄). All other chemicals andsolvents were purchased from Sigma-Aldrich or Fisher Scientific and wereused as supplied without further purification.

Optical rotations were measured on an ADP 220 polarimeter (BellinghamStanley Ltd.) and concentrations (c) are given in g/100 mL. Meltingpoints were measured using a digital melting point apparatus(Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum1000 FT IR Spectrometer. ¹H and ¹³C NMR spectra were acquired at 300 Kusing a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively.Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signalsare designated as s (singlet), d (doublet), t (triplet), dt (doubletriplet), dd (doublet of doublets), ddd (double doublet of doublets) orm (multiplet), with coupling constants given in Hertz (Hz). Massspectroscopy (MS) data were collected using a Waters Micromass ZQinstrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. WatersMicromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35;Extractor (V), 3.0; Source temperature (° C.), 100; DesolvationTemperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flowrate (L/h), 250. High-resolution mass spectroscopy (HRMS) data wererecorded on a Waters Micromass QTOF Global in positive W-mode usingmetal-coated borosilicate glass tips to introduce the samples into theinstrument. Thin Layer Chromatography (TLC) was performed on silica gelaluminium plates (Merck 60, F₂₅₄), and flash chromatography utilisedsilica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt(NovaBiochem) and solid-supported reagents (Argonaut), all otherchemicals and solvents were purchased from Sigma-Aldrich and were usedas supplied without further purification. Anhydrous solvents wereprepared by distillation under a dry nitrogen atmosphere in the presenceof an appropriate drying agent, and were stored over 4 Å molecularsieves or sodium wire. Petroleum ether refers to the fraction boiling at40-60° C.

General LC/MS conditions: The HPLC (Waters Alliance 2695) was run usinga mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B)(formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then5% B to 95% B within 3 min. The composition was held for 0.5 min at 95%B, and then returned to 5% B in 0.3 minutes. Total gradient run timeequals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero deadvolume tee piece which passes into the mass spectrometer. Wavelengthdetection range: 220 to 400 nm. Function type: diode array (535 scans).Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm

Example 3

(i) Key Intermediates

(a)

(a-i) (S)-2-(allyloxycarbonylamino)-3-methylbutanoic acid (12)

Allyl chloroformate (36.2 ml, 340.59 mmol, 1.2 eq) was added dropwise toa stirred solution of L-valine (11)(33.25 g, 283.82 mmol, 1.0 eq) andpotassium carbonate (59.27 g, 425.74 mmol, 1.5 eq) in water (650 mL) andTHF (650 mL). The reaction mixture was stirred at room temperature for18 hours, then the solvent was concentrated under reduced pressure andthe remaining solution extracted with diethyl ether (3×100 mL). Theaqueous portion was acidified to pH 2 with conc. HCl and extracted withDCM (3×100 mL). The combined organics were washed with brine, dried overMgSO₄, filtered and concentrated under reduced pressure to afford theproduct as a colourless oil (57.1 g, assumed 100% yield). LC/MS (1.966min (ES⁺)), m/z: 202.1 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.57 (br s,1H), 7.43 (d, 1H, J=8.6 Hz), 5.96-5.86 (m, 1H), 5.30 (ddd, 1H, J=17.2,3.4, 1.7 Hz), 5.18 (ddd, 1H, J=10.4, 2.9, 1.6 Hz), 4.48 (dt, 2H, J=5.3,1.5 Hz), 3.85 (dd, 1H, J=8.6, 6.0 Hz), 2.03 (oct, 1H, J=6.6 Hz), 0.89(d, 3H, J=6.4 Hz), 0.87 (d, 3H, J=6.5 Hz).

(a-ii) (S)-2,5-dioxopyrrolidin-1-yl2-(allyloxycarbonylamino)-3-methylbutanoate (I3)

To a stirred solution of the protected acid I2 (60.6 g, 301.16 mmol, 1.0eq) and N-hydroxysuccinimide (34.66 g, 301.16 mmol, 1.0 eq) in dry THF(800 mL) was added dicyclohexylcarbodiimide (62.14 g, 301.16 mmol, 1eq). The reaction was stirred for 18 hours at room temperature. Thereaction mixture was then filtered, the solid washed with THF and thecombined filtrate was concentrated under reduced pressure. The residuewas re-dissolved in DCM and left to stand at 0° C. for 30 minutes. Thesuspension was filtered and washed with cold DCM. Concentration of thefiltrate under reduced pressure afforded the product as a viscouscolourless oil (84.7 g, assumed 100% yield) which was used in the nextstep without further purification. LC/MS (2.194 min (ES⁺)), m/z: 321.0[M+Na]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.0 (d, 1H, J=8.3 Hz), 5.97-5.87(m, 1H), 5.30 (ddd, 1H, J=17.2, 3.0, 1.7 Hz), 5.19 (ddd, 1H, J=10.4,2.7, 1.4 Hz), 4.52 (dt, 2H, J=5.3, 1.4 Hz), 4.32 (dd, 1H, J=8.3, 6.6Hz), 2.81 (m, 4H), 2.18 (oct, 1H, J=6.7 Hz), 1.00 (d, 6H, J=6.8 Hz),

(a-iii)(S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanoic acid(I4)

A solution of succinimide ester I3(12.99 g, 43.55 mmol, 1.0 eq) in THF(50 mL) was added to a solution of L-alanine (4.07 g, 45.73 mmol, 1.05eq) and NaHCO₃ (4.02 g, 47.90 mmol, 1.1 eq) in THF (100 mL) and H₂O (100mL). The mixture was stirred at room temperature for 72 hours when theTHF was removed under reduced pressure. The pH was adjusted to 3-4 withcitric acid to precipitate a white gum. After extraction with ethylacetate (6×150 mL), the combined organics were washed with H₂O (200 mL),dried over MgSO₄, filtered and concentrated under reduced pressure.Trituration with diethyl ether afforded the product as a white powderwhich was collected by filtration and washed with diethyl ether (5.78 g,49%). LC/MS (1.925 min (ES⁺)), m/z: 273.1 [M+H]⁺. ¹H NMR (400 MHz,DMSO-d₆) δ 12.47 (br s, 1H), 8.17 (d, 1H, J=6.8 Hz), 7.16 (d, 1H, J=9.0Hz), 5.95-5.85 (m, 1H), 5.29 (dd, 1H, J=17.2, 1.7 Hz), 5.17 (dd, 1H,J=10.4, 1.5 Hz), 4.46 (m, 2H), 4.18 (quin, 1H, J=7.2 Hz), 3.87 (dd, 1H,J=9.0, 7.1 Hz), 1.95 (oct, 1H, J=6.8 Hz), 1.26 (d, 3H, J=7.3 Hz), 0.88(d, 3H, J=6.8 Hz), 0.83 (d, 3H, J=6.8 Hz).

(a-iv) Allyl(S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-1-oxopropan-2-ylamino)-3-methyl-1-oxobutan-2-ylcarbamate(I5)

EEDQ (5.51 g, 22.29 mmol, 1.05 eq) was added to a solution ofp-aminobenzyl alcohol (2.74 g, 22.29 mmol, 1.05 eq) and acid I4 (5.78 g,21.23 mmol, 1 eq) in dry THF (100 mL). and stirred at room temperaturefor 72 hours. The reaction mixture was then concentrated under reducedpressure and the resulting brown solid was triturated with diethyl etherand filtered with subsequent washing with an excess of diethyl ether toafford the product as an off-white solid (7.1 g, 88%). LC/MS (1.980 min(ES⁺)), m/z: 378.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.89 (br s, 1H),8.13 (d, 1H, J=7.0 Hz), 7.52 (d, 2H, J=8.5 Hz), 7.26 (m, 1H), 7.23 (d,2H, J=8.5 Hz), 5.91 (m, 1H), 5.30 (m, 1H), 5.17 (m, 1H), 4.46 (m, 2H),5.09 (t, 1H, J=5.6 Hz), 4.48 (m, 2H), 4.42 (m, 3H), 3.89 (dd, 1H, J=8.6,6.8 Hz), 1.97 (m, 1H), 1.30 (d, 3H, J=7.1 Hz), 0.88 (d, 3H, J=6.8 Hz),0.83 (d, 3H, J=6.7 Hz).

(b)

1-iodo-2-oxo-6, 9, 12,15-tetraoxa-3-azaoctadecan-18-oic acid (I7)

A solution of iodoacetic anhydride (0.250 g, 0.706 mmol, 1.1 eq) in dryDCM (1 mL) was added to amino-PEG₍₄₎-acid I6 (0.170 g, 0.642 mmol, 1.0eq) in DCM (1 mL). The mixture was stirred in the dark at roomtemperature overnight. The reaction mixture was washed with 0.1 M HCl,water, dried over MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by flash chromatography (silica gel,3% MeOH and 0.1% formic acid in chloroform to 10% MeOH and 0.1% formicacid in chloroform) to afford the product as an orange oil (0.118 g,42%). LC/MS (1.623 min (ES⁺)), m/z: 433.98 [M+H]⁺. ¹H NMR (400 MHz,CDCl₃) δ 8.069 (s, 1H), 7.22 (br s, 1H), 3.79 (t, 2H, J=5.8 Hz), 3.74(s, 2H), 3.72-3.58 (m, 14H), 3.50-3.46 (m, 2H), 2.62 (t, 2H, J=5.8 Hz).

(ii) (11 S,11 aS)-allyl11-(tert-butyldimethylsilyloxy)-8-(3-iodopropoxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(34)

(a)(S)-5-((tert-butyldimethylsilyloxy)methyl)-1-(5-methoxy-2-nitro-4-(triisopropylsilyloxy)benzoyl)-4,5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate (7)

Triflic anhydride (28.4 g, 100.0 mmol, 3.0 eq) was added dropwise, over25 mins, to a vigorously stirred solution of the ketone 6 (19.5 g, 30.0mmol, 1.0 eq) in DCM (550 mL) containing 2,6-lutidine (14.4 g, 130.0mmol, 4.0 eq) at −50° C. The reaction mixture was stirred for 1.5 hourswhen LC/MS indicated complete reaction. The organic phase was washedsuccessively with water (100 mL), saturated sodium bicarbonate (150 mL),brine (50 mL), and the organic phase was dried over MgSO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography (silica gel, 90/10 v/v n-hexane/EtOAc) to afford theproduct as a pale yellow oil (19.5 g, 82%). LC/MS (4.391 min (ES⁺)),m/z: 713.25 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) d 7.68 (s, 1H), 6.72 (s,1H), 6.02 (t, 1H, J=1.9 Hz), 4.75 (m, 1H), 4.05 (m, 2H), 3.87 (s, 3H),3.15 (ddd, 1H, J=16.2, 10.3, 2.3 Hz), 2.96 (ddd, 1H, J=16.2, 4.0, 1.6Hz), 1.28-1.21 (m, 3H), 1.07 (d, 18H, J=7.2 Hz), 0.88 (s, 9H), 0.09 (s,3H), 0.08 (s, 3H).

(b)(S,E)-(2-((tert-butyldimethylsilyloxy)methyl)-4-(prop-1-enyl)-2,3-dihydro-1H-pyrrol-1-yl)(5-methoxy-2-nitro-4-(triisopropylsilyloxy)phenyl)methanone(27)

Tetrakis(triphenylphosphine)palladium(0) (0.41 g, 0.35 mmol, 0.03 eq)was added to a mixture of the triflate 7 (8.4 g, 11.8 mmol, 1.0 eq),E-1-propene-1-ylboronic acid (1.42 g, 16.5 mmol, 1.4 eq) and potassiumphosphate (5.0 g, 23.6 mmol, 2.0 eq) in dry dioxane (60 mL) under anitrogen atmosphere. The mixture was stirred at 25° C. for 120 mins whenLC/MS indicated complete reaction. Ethyl acetate (120 mL) and water (120mL) were added, the organic phase was removed, washed with brine (20mL), dried over MgSO₄, filtered and concentrated under reduced pressure.The residue was purified by flash chromatography (silica gel, 95/5 v/vn-hexane/EtOAc to 90/10 v/v n-hexane/EtOAc) to afford the product as ayellow foam (4.96 g, 70%). LC/MS (4.477 min (ES⁺)), m/z: 605.0 [M+H]⁺.¹H NMR (400 MHz, CDCl₃) δ 7.67 (s, 1H), 6.74 (s, 1H), 5.93 (d, 1H,J=15.4 Hz), 5.67 (s, 1H), 4.65 (m, 1H), 4.04 (m, 2H), 3.86 (s, 3H), 2.85(m, 1H), 2.71 (m, 1H), 1.72 (dd, 3H, J=6.8, 1.0 Hz), 1.30-1.22 (m, 3H),1.07 (d, 18H, J=7.2 Hz), 0.87 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H).

(c)(S,E)-(2-amino-5-methoxy-4-(triisopropylsilyloxy)phenyl)(2-((tert-butyldimethylsilyloxy)methyl)-4-(prop-1-enyl)-2,3-dihydro-1H-pyrrol-1-yl)methanone(28)

Zinc dust (22.0 g, 0.33 mol, 37 eq) was added, in portions over 20 mins,to a solution of the propenyl intermediate 27 (5.5 g, 9.1 mmol, 1.0 eq)in 5% v/v formic acid/ethanol (55 mL), using an ice bath to maintain thetemperature between 25-30° C. After 30 mins, the reaction mixture wasfiltered through a short bed of Celite®. The Celite® was washed withethyl acetate (65 mL) and the combined organics were washed successivelywith water (35 mL), saturated sodium bicarbonate (35 mL) and brine (10mL). The organic phase was dried over MgSO₄, filtered and concentratedunder reduced pressure. The residue was purified by flash chromatography(silica gel, 90/10 v/v n-hexane/EtOAc) to afford the product as a paleyellow oil (3.6 g, 69.0%). LC/MS (4.439 min (ES⁺)), m/z: 575.2 [M+H]⁺.¹H NMR (400 MHz, CDCl₃) δ 6.75 (m, 1H), 6.40 (br s, 1H), 6.28 (m, 1H),6.11 (d, 1H, J=15.4 Hz), 5.53 (m, 1H), 4.67 (m, 1H), 4.36 (m, 2H), 3.93(br s, 1H), 3.84 (br s, 1H), 3.73 (s, 3H), 2.86 (dd, 1H, J=15.7, 10.4Hz), 2.73 (dd, 1H, J=15.9, 4.5 Hz), 1.80 (dd, 3H, J=6.8, 1.3 Hz),1.35-1.23 (m, 3H), 1.12 (d, 18H, J=7.3 Hz), 0.89 (s, 9H), 0.08 (s, 3H),0.07 (s, 3H).

(d) (S,E)-allyl2-(2-((tert-butyldimethylsilyloxy)methyl)-4-(prop-1-enyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-(triisopropylsilyloxy)phenylcarbamate(29)

Allyl chloroformate (0.83 g, 6.88 mmol, 1.1 eq) was added to a solutionof the amine 28 (3.6 g, 6.26 mmol, 1.0 eq) in dry DCM (80 mL) containingdry pyridine (1.09 g, 13.77 mmol, 2.2 eq) at −78° C. The dry ice wasremoved and the reaction mixture allowed to warm to room temperature.After stirring for a further 15 minutes, LC/MS indicated completereaction. The organic phase was washed successively with 0.01N HCl (50mL), saturated sodium bicarbonate (50 mL), brine (10 mL), dried overMgSO₄, filtered and concentrated under reduced pressure to leave a paleyellow oil which was used in the next step without further purification(4.12 g, assumed 100% yield). LC/MS (4.862 min (ES⁺)), m/z: 659.2[M+H]⁺.

(e) (S,E)-allyl2-(2-(hydroxymethyl)-4-(prop-1-enyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-(triisopropylsilyloxy)phenylcarbamate(30)

The crude intermediate 29 (assumed 100% yield, 4.12 g, 6.25 mmol, 1.0eq) was dissolved in a mixture of acetic acid (70 mL), methanol (10 mL),THF (10 mL) and water (20 mL) and allowed to stir at room temperature.After 6 hours the reaction mixture was diluted with ethyl acetate (500mL) and washed successively with water (2×500 mL), saturated sodiumbicarbonate (300 mL) and brine (50 mL). The organic phase was dried overMgSO₄, filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, 1/99 v/v methanol/DCM to5/95 v/v methanol/DCM) to afford the product as a yellow oil and afurther 1 g of unreacted starting material was recovered. This materialwas subjected to the same reaction conditions as above, but was leftstirring for 16 h. After work up and purification, additional productwas isolated (2.7 g, 79%, 2 steps) LC/MS (3.742 min (ES⁺)), m/z: 545.2[M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.38 (m, 1H), 7.72 (m, 1H), 6.81 (s,1H), 6.37 (m, 1H), 6.10 (d, 1H, J=15.8 Hz), 5.97 (m, 1H), 5.53 (m, 1H),5.36 (ddd, 1H, J=17.2, 3.1, 1.5 Hz), 5.25 (ddd, 1H, J=10.4, 2.5, 1.3Hz), 4.78 (m, 1H), 4.65 (dt, 2H, J=5.7, 1.3 Hz), 3.84 (m, 3H), 3.79 (s,3H), 3.04 (dd, 1H, J=16.7, 10.5 Hz), 2.40 (dd, 1H, J=16.0, 4.5 Hz), 1.82(dd, 3H, J=6.8, 1.0 Hz), 1.36-1.26 (m, 3H), 1.14 (d, 18H, J=7.3 Hz).

(f) (11S,11aS)-allyl11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(31)

Dry dimethyl sulfoxide (1.16 g, 14.87 mmol, 3.0 eq) was added dropwiseto a solution of oxalyl chloride (0.94 g, 7.43 mmol, 1.5 eq) in DCM (25mL) at −78° C. under an atmosphere of nitrogen. Maintaining thetemperature at −78° C., after 10 mins a solution of the primary alcohol30 (2.7 g, 4.96 mmol, 1.0 eq) in DCM (20 mL) was added dropwise. After afurther 15 mins, dry triethylamine (2.5 g, 24.78 mmol, 5.0 eq) wasadded, and the reaction mixture allowed to warm to room temperature. Thereaction mixture was washed successively with cold 0.1N HCl (50 mL),saturated sodium hydrogen carbonate (50 mL) and brine (10 mL) and theorganic layer was dried over MgSO₄, filtered and concentrated underreduced pressure to afford the product as a yellow oil which was used inthe next step without further purification (2.68 g, assumed 100% yield).LC/MS (3.548 min (ES⁺)), m/z: 543.2 [M+H]⁺.

(g) (11 S, 1 aS)-allyl11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(32)

Tert-butyldimethylsilyltrifluoromethane sulfonate (3.93 g, 14.87 mmol,3.0 eq) was added to a solution of the carbinolamine 31 (assumed 100%yield, 2.68 g, 4.96 mmol, 1.0 eq) and 2,6-lutidine (2.12 g, 19.83 mmol,4.0 eq) in dry DCM (40 mL) at 0° C. under an atmosphere of nitrogen.After 10 minutes, the reaction mixture was allowed to warm to roomtemperature and stirred for a further 60 minutes. The organic phase waswashed successively with water (10 mL), saturated sodium bicarbonate (10mL) and brine (5 mL), dried over MgSO₄, filtered and concentrated underreduced pressure. The residue was purified by flash chromatography(silica gel, chloroform to 2/98 v/v Methanol/chloroform) to afford theproduct as a yellow oil (2.0 g, 63%, 2 steps). LC/MS (4.748 min (ES⁺)),m/z: 657.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.19 (s, 1H), 6.86 (m, 1H),6.66 (s, 1H), 6.22 (d, 1H, J=15.4 Hz), 5.81 (d, 1H, J=8.8 Hz), 5.78 (m,1H), 5.48 (m, 1H), 5.11 (d, 1H, J=5.0 Hz), 5.08 (m, 1H), 4.58 (dd, 1H,J=13.4, 5.4 Hz), 4.35 (dd, 1H, J=13.2, 5.7 Hz), 3.83 (s, 3H), 3.76 (s,1H), 3.00 (dd, 1H, J=15.6, 11.0 Hz), 2.53 (m, 1H), 1.81 (dd, 3H, J=6.8,0.9 Hz), 1.30-1.18 (m, 3H), 1.08 (d, 9H, J=2.3 Hz), 1.06 (d, 9H, J=2.3Hz), 0.86 (s, 9H), 0.25 (s, 3H), 0.18 (s, 3H).

(h) (11S,11aS)-allyl11-(tert-butyldimethylsilyloxy)-8-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(33)

Lithium acetate dihydrate (0.31 g, 3.04 mmol, 1.0 eq) was added to asolution of the diazepine 32 (2.0 g, 3.04 mmol, 1.0 eq) in wet DMF (20mL) at 25° C. and stirred for 4 hours. The reaction mixture was dilutedwith ethyl acetate (200 mL) and washed successively with 0.1M citricacid (50 mL, pH 3), water (50 mL) and brine (10 mL), dried over MgSO₄,filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, 50/50 v/v n-hexane/EtOActo 25/75 v/v n-hexane/EtOAc) to afford the product as a pale yellowsolid (0.68 g, 45%). LC/MS (3.352 min (ES⁺)), m/z: 501.1 [M+H]⁺. ¹H NMR(400 MHz, CDCl₃) δ 7.02 (s, 1H), 6.66 (m, 1H), 6.53 (s, 1H), 6.03 (d,1H, J=15.5 Hz), 5.80 (s, 1H), 5.63 (d, 1H, J=8.9 Hz), 5.55 (m, 1H), 5.29(m, 1H), 4.87 (m, 2H), 4.39 (dd, 1H, J=13.5, 4.2 Hz), 4.20 (dd, 1H,J=13.2, 5.7 Hz), 3.73 (s, 3H), 3.59 (m, 1H), 2.81 (dd, 1H, J=16.1, 10.5Hz), 2.35 (d, 1H, J=15.7 Hz), 1.61 (d, 3H, J=6.4 Hz), 0.67 (s, 9H), 0.05(s, 3H), 0.00 (s, 3H).

(i) (11S,11aS)-allyl11-(tert-butyldimethylsilyloxy)-8-(3-iodopropoxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(34)

Diiodopropane (0.295 g, 1.00 mmol, 5.0 eq) and potassium carbonate(0.028 g, 0.20 mmol, 1.0 eq) were added to a solution of the phenol 33(0.100 g, 0.020 mmol, 1.0 eq) in dry acetone (5 mL). The reactionmixture was heated at 60° C. for 6 hours when LC/MS showed completereaction. The reaction mixture was concentrated to dryness under reducedpressure and the residue was purified by flash chromatography (silicagel, 75/25 v/v n-hexane/EtOAc to 50/50 v/v n-hexane/EtOAc) to afford theproduct as a colourless oil (0.074 g, 56%). LC/MS (3.853 min (ES⁺)),m/z: 669.0 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.26 (s, 1H), 6.90 (s, 1H),6.68 (s, 1H), 6.24 (d, 1H, J=15.3 Hz), 5.87 (d, 1H, J=8.9 Hz), 5.78 (m,1H), 5.53 (m, 1H), 5.12 (m, 2H), 4.65 (m, 2H), 4.41 (m, 1H), 4.11 (m,1H), 3.93 (s, 3H), 3.81 (m, 1H), 3.40 (t, 2H, J=6.7 Hz), 3.05 (dd, 1H,J=16.3, 10.1 Hz), 2.57 (m, 1H), 2.34 (m, 2H), 1.84 (d, 3H, J=6.6 Hz),0.92 (s, 9H), 0.28 (s, 3H), 0.26 (s, 3H).

(iii) (11 S, 11aS)-4-((S)-((S)-2-(alloxycarbonylamino)-3-methylbutanamido)propanamido)benzyl11-(tert-butyldimethylsilyloxy)-8-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-env)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(39)

(a) Allyl((S)-1-(((S)-1-((4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-((E)-prop-1-en-1-yl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(35)

Triethylamine (0.256 mL, 1.84 mmol, 2.2 eq) was added to a stirredsolution of the amine 28 (0.480 g, 0.835 mmol, 1.0 eq) and triphosgene(0.089 g, 0.301 mmol, 0.36 eq) in dry THF (15 mL) at 5° C. (ice bath).The progress of the isocyanate reaction was monitored by periodicallyremoving aliquots from the reaction mixture and quenching with methanoland performing LCMS analysis. Once the isocyanate reaction was completea solution of Alloc-Val-Ala-PABOH 15 (0.473 g, 1.25 mmol, 1.5 eq) andtriethylamine (0.174 mL, 1.25 mmol, 1.5 eq) in dry THF (10 mL) wasrapidly added by injection to the freshly prepared isocyanate. Thereaction was allowed to stir at 40° C. for 4 hours followed by stirringat room temperature overnight. The mixture was concentrated underreduced pressure, and purified by flash chromatography (silica gel,20/80 v/v n-hexane/EtOAc to 50/50 v/v n-hexane/EtOAc, then 1/99 v/vDCM/MeOH to 5/95 v/v DCM/MeOH) to afford the product as a yellow solid(0.579 g, 71%). LC/MS (4.468 min (ES⁺)), m/z: 978.55 [M+H]⁺. ¹H NMR (400MHz, CDCl₃) δ 8.63 (br s, 1H), 8.42 (s, 1H), 7.78 (br s, 1H), 7.53 (d,2H, J=8.1 Hz), 7.31 (d, 2H, J=8.6 Hz), 6.76 (s, 1H), 6.59 (d, 1H, J=7.6Hz), 6.36 (br s, 1H), 6.04 (d, 1H, J=15.9 Hz), 5.90 (m, 1H), 5.55 (m,1H), 5.33-5.21 (m, 3H), 5.10 (s, 2H), 4.66 (m, 2H), 4.57 (dd, 2H, J=5.6,1.0 Hz), 3.98 (dd, 1H, J=7.3, 6.8 Hz), 3.90 (m, 1H), 3.81 (m, 1H), 3.78(s, 3H), 2.82 (dd, 1H, J=15.4, 9.6 Hz), 2.72 (dd, 1H, J=15.9, 3.5 Hz),2.17 (m, 1H), 1.78 (dd, 3H, J=6.5, 0.8 Hz), 1.46 (d, 3H, J=7.1 Hz), 1.29(m, 3H), 1.11 (d, 18H, J=7.1 Hz), 0.97 (d, 3H, J=6.8 Hz), 0.92 (d, 3H,J=6.8 Hz), 0.83 (s, 9H), 0.04 (s, 3H), 0.01 (s, 3H).

(b) Allyl((S)-1-(((S)-1-((4-((((2-((S)-2-(hydroxymethyl)-4-((E)-prop-1-en-1-yl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (36)

The silyl ether 35 (1.49 g, 1.52 mmol, 1.0 eq) was dissolved in a7:1:1:2 mixture of acetic acid/methanol/tetrahydrofuran/water (14:2:2:4mL) and allowed to stir at room temperature. After 2 hours the reactionwas diluted with EtOAc (100 mL), washed sequentially with water, aq.sodium bicarbonate then brine. The organic phase was then dried overMgSO₄, filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, 100/0 then 99/1 to 92/8v/v DCM/MeOH) to afford the product as an orange solid (1.2 g, 92%).LC/MS (3.649 min (ES⁺)), m/z: 865.44 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ8.44 (s, 1H), 8.35 (s, 1H), 7.69 (br s, 1H), 7.53 (d, 2H, J=8.7 Hz),7.32 (d, 2H, J=8.3 Hz), 6.78 (s, 1H), 6.56 (m, 2H), 6.32 (br s, 1H),6.05 (d, 1H, J=14.9 Hz), 5.90 (m, 1H), 5.56 (m, 1H), 5.30 (m, 2H), 5.22(m, 1H), 5.10 (d, 2H, J=3.1 Hz), 4.73 (m, 1H), 4.64 (m, 1H), 4.57 (d,2H, J=5.8 Hz), 4.01 (m, 1H), 3.79 (m, 2H), 3.76 (s, 3H), 2.98 (dd, 1H,J=16.3, 10.2 Hz), 2.38 (dd, 1H, J=16.6, 4.1 Hz), 2.16 (m, 1H), 1.78 (dd,3H, J=6.8, 0.9 Hz), 1.46 (d, 3H, J=7.1 Hz), 1.29 (m, 3H), 1.11 (d, 18H,J=7.4 Hz), 0.97 (d, 3H, J=6.7 Hz), 0.92 (d, 3H, J=6.8 Hz).

(c)(11S,11aS)-4-((S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyl11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[,2-a][1,4]diazepine-10(5H)-carboxylate (37)

Dry dimethyl sulfoxide (0.180 g, 2.3 mmol, 3.0 eq) was added dropwise toa solution of oxalyl chloride (0.147 g, 1.1 mmol, 1.5 eq) in DCM (10 mL)at −78° C. under an atmosphere of nitrogen. Maintaining the temperatureat −78° C., after 20 minutes, a solution of the primary alcohol 36(0.666 g, 0.77 mmol, 1.0 eq) in DCM (10 mL) was added dropwise. After afurther 15 minutes, dry triethylamine (0.390 g, 3.85 mmol, 5.0 eq) wasadded, and the reaction mixture allowed to warm to room temperature. Thereaction mixture was washed successively with cold 0.1N HCl (10 mL),saturated sodium hydrogen carbonate (10 mL) and brine (5 mL). Theorganic layer was then dried over MgSO₄, filtered and concentrated underreduced pressure. The residue was then purified by flash chromatography(silica gel, 50/50 v/v n-hexane/EtOAc to 25/75 v/v n-hexane/EtOAc) toafford the product as a white solid (0.356 g, 54%). LC/MS (3.487 min(ES⁺)), m/z: 862.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.34 (br s, 1H),7.47 (d, 2H, J=7.6 Hz), 7.17 (s, 1H), 7.14 (d, 2H, J=7.5 Hz), 6.86 (brs, 1H), 6.65 (br s, 1H), 6.42 (d, 1H, J=7.6 Hz), 6.22 (d, 1H, J=14.4Hz), 5.80 (m, 1H), 5.40 (m, 1H), 5.53 (m, 1H), 5.32 (m, 1H), 5.21 (d,2H, J=9.6 Hz), 5.06 (d, 1H, J=12.3 Hz), 4.90 (m, 1H), 4.58 (m, 3H), 3.98(m, 1H), 3.84 (m, 1H), 3.81 (s, 3H), 3.50 (m, 1H), 3.05 (dd, 1H, J=16.0,10.3 Hz), 2.76 (m, 1H), 2.15 (m, 1H), 1.80 (dd, 3H, J=6.7, 0.8 Hz), 1.44(d, 3H, J=7.1 Hz), 1.16 (m, 3H), 1.01 (d, 18H, J=6.6 Hz), 0.96 (d, 3H,J=6.8 Hz), 0.92 (d, 3H, J=6.8 Hz).

(d)(11S,11aS)-4-((S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyl11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][,4]diazepine-10(5H)-carboxylate (38)

Tert-butyldimethylsilyltrifluoromethane sulfonate (0.46 g, 1.74 mmol,3.0 eq) was added to a solution of secondary alcohol 37 (0.5 g, 0.58mmol, 1.0 eq) and 2,6-lutidine (0.25 g, 2.32 mmol, 4.0 eq) in dry DCM(10 mL) at 0° C. under an atmosphere of nitrogen. After 10 minutes, thereaction mixture was allowed to warm to room temperature and stirred fora further 120 mins. The organic phase was then washed successively withwater (10 mL), saturated sodium bicarbonate (10 mL) and brine (5 mL),dried over MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography (silica gel, 50/50 v/vn-hexane/EtOAc) to afford the product as a white solid (0.320 g, 57%).LC/MS (4.415 min (ES⁺)), m/z: 976.52 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ8.31 (br s, 1H), 7.48 (d, 2H, J=8.0 Hz), 7.21 (s, 1H), 7.14 (d, 2H,J=8.3 Hz), 6.89 (s, 1H), 6.65 (s, 1H), 6.38 (d, 1H, J=7.3 Hz), 6.25 (d,1H, J=14.6 Hz), 5.93 (m, 1H), 5.85 (d, 1H, J=8.8 Hz), 5.50 (m, 1H), 5.34(m, 1H), 5.24 (m, 2H), 5.15 (d, 1H, J=12.5 Hz), 4.86 (d, 1H, J=12.2 Hz),4.62 (m, 3H), 4.01 (m, 1H), 3.86 (s, 3H), 3.78 (m, 1H), 3.04 (m, 1H),2.56 (m, 1H), 2.20 (m, 1H), 1.84 (dd, 3H, J=6.6, 0.7 Hz), 1.48 (d, 3H,J=6.8 Hz), 1.20 (m, 3H), 1.05 (d, 9H, J=2.9 Hz), 1.03 (d, 9H, J=2.9 Hz),0.99 (d, 3H, J=6.8 Hz), 0.95 (d, 3H, J=6.8 Hz), 0.88 (s, 9H), 0.27 (s,3H), 0.14 (s, 3H).

(e)(11S,11aS)-4-((S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyl1I-(tert-butyldimethylsilyloxy)-8-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(39)

Lithium acetate dihydrate (0.010 g, 0.10 mmol, 1.0 eq) was added to asolution of the silyl ether 38 (0.100 g, 0.10 mmol, 1.0 eq) in wet DMF(2 mL) at 25° C. for 3 hours. The reaction mixture was then diluted withethyl acetate (20 mL) and washed successively with 0.1M citric acid (20mL, pH 3), water (20 mL) and brine (5 mL), dried over MgSO₄, filteredand concentrated under reduced pressure. The residue was purified byflash chromatography (silica gel, 5/95 v/v methanol/DCM) to afford theproduct as a pale yellow oil (0.070 g, 83%). LC/MS (3.362 min (ES⁺)),m/z: 820.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.39 (s, 1H), 7.48 (d, 2H,J=8.2 Hz), 7.25 (s, 1H), 7.12 (d, 2H, J=8.1 Hz), 6.88 (s, 1H), 6.68 (s,1H), 6.47 (d, 1H, J=7.6 Hz), 6.24 (d, 1H, J=15.2 Hz), 6.03 (s, 1H), 5.92(m, 1H), 5.84 (d, 1H, J=8.9 Hz), 5.50 (m, 1H), 5.34 (m, 1H), 5.26 (m,2H), 5.18 (d, 1H, J=12.3 Hz), 4.80 (d, 1H, J=12.4 Hz), 4.66-4.60 (m,3H), 4.02 (m, 1H), 3.95 (s, 3H), 3.81 (m, 1H), 3.03 (m, 1H), 2.57 (m,1H), 2.19 (m, 1H), 1.84 (dd, 3H, J=6.8, 0.8 Hz), 1.48 (d, 3H, J=7.1 Hz),1.00 (d, 3H, J=6.8 Hz), 0.95 (d, 3H, J=6.8 Hz), 0.87 (s, 9H), 0.26 (s,3H), 0.12 (s, 3H).

(iv) (11 S,11aS)-4-((20S,23S)-1-iodo-20-isopropyl-23-methyl-2,18,21-trioxo-6,9,12,15-tetraoxa-3,19,22-triazatetracosanamido)benzyl11-hydroxy-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(26, A)

(a) (11S,11aS)-allyl8-(3-((11S,11aS)-10-((4-((R)-2-((R)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyloxy)carbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(40)

Potassium carbonate (0.030 g, 0.21 mmol, 1.0 eq) was added to a solutionof the phenol 39 (0.175 g, 0.21 mmol, 1.0 eq) and the iodo linker 34(0.214 g, 0.32 mmol, 1.5 eq) in acetone (10 mL). The reaction mixturewas heated under a nitrogen atmosphere at 75° C. in a sealed flask for17 hours. The reaction mixture was concentrated to dryness under reducedpressure and purified by flash chromatography (silica gel, 2/98 v/vmethanol/DCM to 5/95 v/v methanol/DCM) to afford the product as a paleyellow solid (0.100 g, 35%). LC/MS (4.293 min (ES⁺)), m/z: 1359.13 [M]⁺.

(b) (11S,11aS)-allyl8-(3-((11S,11aS)-10-((4-((R)-2-((R)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyloxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(41)

Tetra-n-butylammonium fluoride (1M, 0.22 mL, 0.22 mmol, 2.0 eq) wasadded to a solution of silyl ether 40 (0.150 g, 0.11 mmol, 1.0 eq) indry THF (2 mL). The reaction mixture was stirred at room temperature for20 minutes, after which LC/MS indicated complete reaction. The reactionmixture was diluted with ethyl acetate (10 mL) and washed sequentiallywith water (5 mL) and brine (5 mL). The organic phase was dried overMgSO₄, filtered and concentrated under reduced pressure to leave ayellow solid. Purification by flash chromatography (silica gel, 6/94 v/vmethanol/DCM to 10/90 v/v methanol/DCM) afforded the product as a paleyellow solid (0.090 g, 73%). LC/MS (2.947 min (ES⁺)), m/z: 1154.0[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.39 (br s, 1H), 7.39 (d, 2H, J=7.6Hz), 7.18 (d, 2H, J=10.6 Hz), 7.10 (m, 3H), 6.86 (d, 2H, J=10.0 Hz),6.74 (s, 1H), 6.55 (s, 1H), 6.22 (dd, 2H, J=15.3, 6.6 Hz), 5.85 (m, 2H),5.74 (m, 3H), 5.52 (m, 2H), 5.22 (m, 1H), 5.00 (m, 2H), 4.57 (m, 6H),4.41 (m, 2H), 4.09 (m, 4H), 3.85 (m, 11H), 3.06 (m, 2H), 2.76 (m, 2H),2.20 (m, 2H), 2.08 (m, 1H), 1.79 (d, 6H, J=6.4 Hz), 1.40 (d, 3H, J=6.1Hz), 0.90 (m, 6H).

(c)(11S,11aS)-4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl11-hydroxy-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(25)

Tetrakis(triphenylphospene)palladium(0) (0.005 g, 0.005 mmol, 0.06 eq)was added to a solution of the bis-carbinolamine 41 (0.090 g, 0.08 mmol,1.0 eq) and pyrrolidine (16 μL, 0.20 mmol, 2.5 eq) in dry DCM (5 mL).After 20 minutes, the reaction mixture was diluted with DCM (10 mL) andwashed sequentially with saturated ammonium chloride (5 mL) and brine (5mL), dried over MgSO₄, filtered and the solvent was removed underreduced pressure to leave the crude product as a pale yellow solid whichwas used in the next step without further purification (0.075 g, assumed100% yield). LC/MS (2.060 min (ES⁺)), m/z: 947.2 [M+H]⁺.

(d) (11S,11aS)-4-((20S,23S)-1-iodo-20-isopropyl-23-methyl-2,18,21-trioxo-6,9,12,15-tetraoxa-3,19,22-triazatetracosanamido)benzyl11-hydroxy-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(26, A)

EDCI (0.015 g, 0.08 mmol, 1.0 eq) was added to a solution of amine 25(assumed 100% yield 0.075 g, 0.08 mmol, 1.0 eq) andiodoacetamide-PEG₄-acid I7 (0.034 g, 0.08 mmol, 1.0 eq) in drydichloromethane (5 mL) and the reaction was stirred in the dark. After50 minutes, a further amount of iodoacetamide-PEG₄-acid I7 (0.007 g,0.016 mmol, 0.2 eq) was added along with a further amount of EDCI (0.003g, 0.016 mmol, 0.2 eq). After a total of 2.5 hours, the reaction mixturewas diluted with dichloromethane (15 mL) and washed sequentially withwater (10 mL) and brine (10 mL). The organic phase was dried over MgSO₄,filtered and concentrated under reduced pressure. The resulting residuewas purified by flash chromatography (silica gel, Chloroform 100% to90:10 v/v Chloroform:Methanol). Pure fractions were combined to affordthe product (0.0254 g, 23%, 2 steps). The crude fractions were collectedand purified by preparative TLC (silica gel, 90:10 v/vChloroform:Methanol) to afford a second batch of product (0.0036 g, 3%,2 steps). LC/MS (2.689 min (ES⁺)), m/z: 681.0 1/2[M+2H]⁺.

Example 4: Activity of Released Compounds

K562 Assay

K562 human chronic myeloid leukaemia cells were maintained in RPM1 1640medium supplemented with 10% fetal calf serum and 2 mM glutamine at 37°C. in a humidified atmosphere containing 5% CO₂ and were incubated witha specified dose of drug for 1 hour or 96 hours at 37° C. in the dark.The incubation was terminated by centrifugation (5 min, 300 g) and thecells were washed once with drug-free medium. Following the appropriatedrug treatment, the cells were transferred to 96-well microtiter plates(10⁴ cells per well, 8 wells per sample). Plates were then kept in thedark at 37° C. in a humidified atmosphere containing 5% CO₂. The assayis based on the ability of viable cells to reduce a yellow solubletetrazolium salt,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT,Aldrich-Sigma), to an insoluble purple formazan precipitate. Followingincubation of the plates for 4 days (to allow control cells to increasein number by approximately 10 fold), 20 μL of MTT solution (5 mg/mL inphosphate-buffered saline) was added to each well and the plates furtherincubated for 5 h. The plates were then centrifuged for 5 min at 300 gand the bulk of the medium pipetted from the cell pellet leaving 10-20μL per well. DMSO (200 μL) was added to each well and the samplesagitated to ensure complete mixing. The optical density was then read ata wavelength of 550 nm on a Titertek Multiscan ELISA plate reader, and adose-response curve was constructed. For each curve, an IC₅₀ value wasread as the dose required to reduce the final optical density to 50% ofthe control value.

Compound RelB has an IC₅₀ of 0.425 nM in this assay.

Example 5

Biotin-A20FMD V-Cys-2 (43)

A solution of the peptide 42 (12.06 mg, 4.8 μmol, 1.0 eq) in 1/1acetonitrile/water (1 mL) was added to a solution of 24 (12.06 mg, 4.8μmol, 1.0 eq) in 1/1 acetonitrile/water (1 mL). The solution was stirredat room temperature for 24 hours. The acetonitrile/water was removed bylyophilisation to give a yellow foam. Purification by preparative HPLCfollowed by lyophilisation gave the product as a white foam (7.4 mg,38%)

Preparative HPLC Method:

Reverse-phase ultra-high-performance liquid chromatography (UPLC) wascarried out on Phenomenex Gemini NX 5μ C-18 columns of the followingdimensions: 150×4.6 mm for analysis, and 150×21.20 mm for preparativework. All UPLC experiments were performed with gradient conditions:initial fixed composition 13% B to 75% B over 15 min, held for 2.0 minat 75% B, then 75% B to 13% B within 0.10 min held at 13% for 2.90 min.Total duration of gradient run was 20.00 min. Eluents used were solventA (H₂O with 0.1% Formic acid) and solvent B (CH₃CN with 0.1% Formicacid). Flow rates used were 1.0 ml/min for analytical, and 20.0 ml/minfor preparative HPLC. Detection was at 254 and 280 nm.

Analytical Data: LCMS 1.17 min (ES+) m/z (relative intensity)1330([M+2]⁺/3, 5%); 998([M+3]⁺/4, 70); 798([M+4]⁺/5, 100); 665([M+5]⁺/6,20).

Example 6: Formation of Conjugates

General Antibody Conjugation Procedure

Antibodies are diluted to 1-5 mg/mL in a reduction buffer (examples:phosphate buffered saline PBS, histidine buffer, sodium borate buffer,TRIS buffer). A freshly prepared solution of TCEP(tris(2-carboxyethyl)phosphine hydrochloride) is added to selectivelyreduce cysteine disulfide bridges. The amount of TCEP is proportional tothe target level of reduction, within 1 to 4 molar equivalents perantibody, generating 2 to 8 reactive thiols. After reduction for severalhours at 37° C., the mixture is cooled down to room temperature andexcess drug-linker (A, B) added as a diluted DMSO solution (final DMSOcontent of up to 10% volume/volume of reaction mixture). The mixture isgently shaken at either 4° C. or room temperature for the appropriatetime, generally 1-3 hours for compound B and 10-30 hours for compound A.Excess reactive thiols can be reacted with a ‘thiol capping reagent’like N-ethyl maleimide (NEM) at the end of the conjugation.Antibody-drug conjugates are concentrated using centrifugal spin-filterswith a molecular weight cut-off of 10 kDa or higher, then purified bytangential flow filtration (TFF) or Fast Protein Liquid Chromatography(FPLC). Corresponding antibody-drug conjugates can be determined byanalysis by High-Performance Liquid Chromatography (HPLC) orUltra-High-Performance Liquid Chromatography (UHPLC) to assessdrug-per-antibody ratio (DAR) using reverse-phase chromatography (RP) orHydrophobic-Interaction Chromatography (HIC), coupled with UV-Visible,Fluorescence or Mass-Spectrometer detection; aggregate level and monomerpurity can be analysed by HPLC or UHPLC using size-exclusionchromatography coupled with UV-Visible, Fluorescence orMass-Spectrometer detection. Final conjugate concentration is determinedby a combination of spectroscopic (absorbance at 280, 214 and 330 nm)and biochemical assay (bicinchonic acid assay BCA; Smith, P. K., et al.(1985) Anal. Biochem. 150 (1): 76-85; using a known-concentration IgGantibody as reference). Antibody-drug conjugates are generally sterilefiltered using 0.2 m filters under aseptic conditions, and stored at +4°C., −20° C. or −80° C.

Examples of particular conjugations are described below.

ADC1A

Antibody 1 (15 mg, 102 nanomoles) is diluted into 12.0 mL of a reductionbuffer containing 10 mM sodium borate pH 8.4, 2.5 mM EDTA and a finalantibody concentration of 1.25 mg/mL. A 10 mM solution of TCEP is added(2 molar equivalent/antibody, 204 nanomoles, 20 μL) and the reductionmixture is heated at 37° C. for two hour in an orbital incubator. Aftercooling down to room temperature, compound A is added as a DMSO solution(5 molar equivalent/antibody, 510 nanomoles, in 1.2 mL DMSO). Thesolution is mixed 18 hour at room temperature, then transferred into a15 mL Amicon Ultracell 50 kDa MWCO spin filter, concentrated to ca. 2.0mL and injected into a AKTA™FPLC using a GE Healthcare XK16/70 columnpacked with Superdex 200 PG, eluting with 1.5 mL/min of sterile-filteredPhosphate-buffered saline (PBS). Fractions corresponding to the desiredADC monomer peak are pooled, analysed and sterile-filtered. BCA assaygives a concentration of final ADC1A at 0.67 mg/mL in 12.5 mL, and theobtained mass is 8.4 mg (56% yield). HPLC analysis on a Waters Alliancesystem using a Agilent PLRP-S 1000 A 8 um 150×2.1 mm column eluting witha gradient of water and acetonitrile on a reduced sample of ADC1A at 280nm shows a mixture of light and heavy chains attached to severalmolecules of A, consistent with a drug-per-antibody ratio (DAR) of 2.2molecules of A per antibody. SEC analysis on an AKTA™FPLC using a GEHealthcare XK16/70 column packed with Superdex 200 PG, eluting withsterile-filtered Phosphate-buffered saline (PBS) on a sample of ADC1A at280 nm shows a monomer purity of 95.3% with 4.7% aggregates.

ADC1B

Antibody 1(15 mg, 102 nanomoles) is diluted into 12.0 mL of a reductionbuffer containing 10 mM sodium borate pH 8.4, 2.5 mM EDTA and a finalantibody concentration of 1.25 mg/mL. A 10 mM solution of TCEP is added(2 molar equivalent/antibody, 204 nanomoles, 20 μL) and the reductionmixture was heated at 37° C. for two hour in an orbital incubator. Aftercooling down to room temperature, Compound B is added as a DMSO solution(5 molar equivalent/antibody, 510 nanomoles, in 1.2 mL DMSO). Thesolution is mixed 5 hour at room temperature, then a second amount ofCompound B is added as a DMSO solution (2 molar equivalent/antibody, 200nanomoles, in 0.6 mL DMSO). The solution is mixed 15 hour at roomtemperature, then transferred into a 15 mL Amicon Ultracell 50 kDa MWCOspin filter, concentrated to ca. 2.0 mL and injected into an AKTA™FPLCusing a GE Healthcare XK16/70 column packed with Superdex 200 PG,eluting with 1.5 mL/min of sterile-filtered Phosphate-buffered saline(PBS). Fractions corresponding to ADC1B monomer peak are pooled,analysed and sterile-filtered. BCA assay gives a concentration of finalADC1B at 1.02 mg/mL in 10.5 mL, and the obtained mass is 10.7 mg (71%yield). HPLC analysis on a Waters Alliance system using a Agilent PLRP-S1000 A 8 um 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of ADC1B at 280 nm and 330 nm(drug-linker specific) shows a mixture of light and heavy chainsattached to several molecules of B, consistent with a drug-per-antibodyratio (DAR) of 2.2 molecules of B per antibody. SEC analysis on aAKTA™FPLC using a GE Healthcare XK16/70 column packed with Superdex 200PG, eluting with sterile-filtered Phosphate-buffered saline (PBS) on asample of ADC1B at 280 nm shows a monomer purity of 94.9% with 5.1%aggregates.

Example 7: In Vivo ADC Efficacy Studies

CB.17 SCID mice, aged 8-12 weeks, are injected with 1 mm³ tumourfragments sub cutaneously in the flank. When tumours reach an averagesize of 100-150 mm³, treatment is begun. Mice are weighed twice a week.Tumour size is measured twice a week. Animals are monitoredindividually. The endpoint of the experiment is a tumour volume of 1000mm³ or 60 days, whichever comes first. Responders can be followedlonger.

Groups of 10 xenografted mice are injected i.v. with 0.2 ml of antibodydrug conjugate (ADC), or naked antibody, in phosphate buffered saline(vehicle) or with 0.2 ml of vehicle alone. The concentration of ADC isadjusted to give, for example, 0.3 or 1.0 mg ADC/kg body weight in asingle dose. Three identical doses may be given to each mouse atintervals of, for example, 1 week.

All documents and other references mentioned above are hereinincorporated by reference.

Abbreviations

-   Ac acetyl-   Acm acetamidomethyl-   Alloc allyloxycarbonyl-   Boc di-tert-butyl dicarbonate-   t-Bu tert-butyl-   Bzl benzyl, where Bzl-OMe is methoxybenzyl and Bzl-Me is    methylbenzene-   Cbz or Z benzyloxy-carbonyl, where Z—Cl and Z—Br are chloro- and    bromobenzyloxy carbonyl respectively-   DMF N,N-dimethylformamide-   Dnp dinitrophenyl-   DTT dithiothreitol-   Fmoc 9H-fluoren-9-ylmethoxycarbonyl-   imp N-10 imine protecting group:    3-(2-methoxyethoxy)propanoate-Val-Ala-PAB-   MC-OSu maleimidocaproyl-O—N-succinimide-   Moc methoxycarbonyl-   MP maleimidopropanamide-   Mtr 4-methoxy-2,3,6-trimethtylbenzenesulfonyl-   PAB para-aminobenzyloxycarbonyl-   PEG ethyleneoxy-   PNZ p-nitrobenzyl carbamate-   Psec 2-(phenylsulfonyl)ethoxycarbonyl-   TBDMS tert-butyldimethylsilyl-   TBDPS tert-butyldiphenylsilyl-   Teoc 2-(trimethylsilyl)ethoxycarbonyl-   Tos tosyl-   Troc 2,2,2-trichlorethoxycarbonyl chloride-   Trt trityl-   Xan xanthyl

The invention claimed is:
 1. Compound RelB:

or a pharmaceutically acceptable salt thereof.